DOI: 10.5061/dryad.vx0k6dk0f
title: Devastating disease can cause increased breeding effort and success that improves population resilience

description: Novel and invasive diseases are a key threat to wildlife and can cause
 massive, unprecedented declines and extinctions. The amphibian fungal
 disease chytridiomycosis has had devastating global impacts, but after
 severe declines some amphibian species can persist and even rebound.
 Understanding how these species survive is critical to discovering
 management techniques for supporting declining species. Here we explored
 the impacts of disease on reproduction in frogs, investigating its effect
 on primary and secondary sexual characteristics and breeding effort and
 success.  Male frogs are increasing various facets of their
 breeding efforts resulting in increased offspring. Infected male frogs
 have 1) increased vocal sac coloration, 2) increased sperm quality, and 3)
 higher mating success and father more egg masses than uninfected males.
 Ongoing high mortality due to chytridiomycosis in these species appears to
 be balanced by high reproduction. Management efforts should target the
 natural mechanisms (e.g., breeding) that species use to overcome key
 threats because they are more likely to succeed and be sustainable.
description: Study species <em>Litoria
 verreauxii alpina</em>, the alpine tree frog, is endemic to the
 Australian Alps and is considered critically endangered in both New South
 Wales and Victoria, Australia. Since the introduction of the fungal
 pathogen <em>Batrachochytrium dendrobatidis</em> in the 1980s,
 this once widespread species has experienced dramatic declines and is now
 present in &lt;20% of its former range (Osborne et al. 1999). The
 species now inhabits 8-10 sites across the Alps. While the species is
 inhabiting a much smaller area than they were historically, the
 populations at these remaining sites appear to be relatively stable. The
 species is highly susceptible to B. dendrobatidis infection and
 experiences near complete population turnover every year (Brannelly et al.
 2015, Scheele et al. 2015). It is well understood that the species is
 persisting in these habitats due to breeding (Brannelly et al. 2020a,
 2021a), but it was unclear if the species was actively increasing their
 breeding output to persist or if their baseline reproductive effort was
 high enough to maintain the populations. However, since the introduction
 of B. dendrobatidis to the populations, there has been changes in baseline
 spermatogenesis, indicating that the populations with endemic disease are
 evolving increased reproduction (Brannelly et al. 2021b).
   Secondary Sexual
 Characteristics Clinical infection
 trial   Adult male
 <em>L. v. alpina</em> were bred in captivity at the Melbourne
 Veterinary School (Brannelly et al. 2023). Males were 1 year old and
 sexually mature at the time of the experiment, and randomly selected from
 three clutches of origin, and evenly allocated to each treatment group (n
 = 30, 15 infected and 15 control; block randomisation across clutch and
 treatment). Frogs were housed individually in terraria (34cm x 20cm x
 15cm) on a gravel and moss substrate at 16-19ºC, 12:12hr light:dark cycle
 during the Austral spring, which is their natural breeding season.
 Terraria were flushed daily with carbon-filtered water and were fed
 crickets dusted with vitamin powder (Repashy) twice per week for the
 duration of the experiment.  
 Males were inoculated with a known virulent strain of
 <em>B. dendrobatidis</em> from New South Wales Victoria,
 AUS#46 (WastePoint-L.verreauxii #5-2013-LB.RW). Bd was grown on agar and
 tryptone, gelatin hydrolysate, lactose plates and incubated for three days
 at 20°C. The plates were flooded with 3ml of sterile Milli-Q water and
 agitated for 10 minutes to allow the Bd zoosporangia to release zoospores.
 The inoculum was poured off the plates and the Bd zoospore concentration
 was determined were counted with a haemocytometer. <em>Litoria v.
 alpina</em> males in the Bd treatment group were inoculated with
 5×105 zoospores in 5ml of inoculum. The inoculum was dripped onto their
 dorsum with a syringe and allowed to pool in the 20ml inoculation
 container. The control frogs were given a mock inoculation by flooding
 sterile agar plates with Milli-Q water, and then dripping 5ml of the
 control inoculant onto the animals. All frogs were left in inoculation
 containers for 12 hours, and then returned to their individual terraria
 with gravel and moss substrate.   
 Frogs were swabbed for <em>B. dendrobatidis</em>
 (see methods below), measured, and analysed for breeding colouration
 weekly. The snout-to-vent length (SVL) to the nearest 0.1mm was measured
 using dial callipers, and each frog was weighed to the nearest 0.01g using
 digital laboratory scales. Colour readings were taken using a portable
 spectrophotometer (see methods below) for three measurements on the
 throat, dorsum, and venter. The experiment ended after 4 weeks when some
 animals began to show clinical signs of chytridiomycosis (e.g., anorexia,
 irregular skin slough, splayed leg posture).
   Batrachochytrium dendrobatidis
 testing To collect a sample for <em>B.
 dendrobatidis </em>from each frog, a dry sterile rayon swab (MW-113,
 Medical Wire and Equipment Wiltshire, UK) was rolled five times along each
 of: the ventrolateral surface of the abdomen, the ventral surface of all
 limbs and digits, and the medial surface of the thighs. The tip of the
 swab was then broken off into a 1.5 mL Eppendorf tube and stored at -20ºC
 until processing (approximately 3 months). Skin swab samples were
 extracted using PrepMan Ultra (Thermo Fisher Scientific). The extraction
 method followed the manufacturer’s directions which included: adding 50 μL
 of PrepMan Ultra and 30-40 mg of 0.5 mm silica beads (BioSpec) to each,
 homogenizing samples (using a cell homogenizer) for 2 min at 1400
 oscillations per sec, incubating samples at 95 °C to lyse the cells for 10
 min, and collecting and diluting the supernatant 6:100 in ultra pure water
 before directly analysing for pathogen presence and quantity using qPCR
 (Brannelly et al. 2020b). The remaining extracted DNA was stored at 4 °C.
 With every extraction performed, one <em>B. dendrobatidis</em>
 positive control sample (zoospores from culture) and one negative control
 (swab only) were extracted.  
 We used qPCR (Rotogene, Qiagen) to amplify and quantify the
 <em>B. dendrobatidis</em> DNA in each sample following Boyle
 et al. (2004) with modifications (Jadwani-Bungar et al. n.d., Brannelly et
 al. 2020b). We used a 15 μL reaction volume, with 5μL of template DNA, 7.5
 μL of lo-ROX 2x master mix (SensiFast, Bioline), and a final reaction
 concentration of 900 nM ITS1-3 (Purification: RP-Cartridge Gold, Sequence:
 CCTTGATATAATACAGTGTGCCATATGTC, Eurogentec, Integrated Science), 900 nM
 5.8S Chytr (Purification: RP-Cartridge Gold, Sequence:
 AGCCAAGAGATCCGTTGTCAAA, Eurogenetec, Integrated Science), 250 nM Chytr
 MGB2 5’ 6-FAM-labelled probe (5' 6-FAM - CGA-GTC-GAA-CAA-AAT -
 MGB-Eclipse® 3'; Integrated Science) and 400 ng/μL bovine serum
 albumin (Fisher Biotechologies). The amplification conditions were 2 min
 at 50°C, 10 min at 95°C, followed by 15 s at 95°C and 1 min at 60°C for 40
 cycles. On each qPCR reaction plate we included a series of seven
 plasmid-based Bd standards (purchased from Pisces Molecular, containing
 4.2, 42, 420, 4,200, 42,000, 420,000, and 4,200,000 DNA copies per
 reaction). On each qPCR reaction plate we included a no template control,
 where ultrapure water replaced the template DNA. The extraction positive
 and negative control samples were analysed via qPCR like all sample
 extracted DNA.    DNA copies
 of the <em>B. dendrobatidis </em>ITS gene extracted from each
 swab were estimated by extrapolating the gene copies detected by qPCR
 (Roto-Gene Q 2.3.5 software), considering the elution volume and dilution
 of template DNA. Any sample was determined to be Bd positive if the
 reaction well was amplified, representing at least 2 ITS DNA copies
 detected in the sample. If the sample was considered negative for Bd, the
 Bd DNA copies were coded as 0 for that sample (Brannelly et al. 2020b).
 Samples were analysed via qPCR in singlicate, which is a common and
 accepted practice for chytrid infection studies (Brannelly et al. 2015,
 2017). All positive extraction controls were positive, all negative
 extraction controls were negative, and all no-template qPCR controls were
 negative for <em>B. dendrobatidis</em> DNA
 copies.    Skin colour
 methodology Colour readings were taken using a
 portable spectrophotometer (OceanOptics USB2000+), probe (OceanOptics
 R400-UV-VIS), and light source (OceanOptics PX-2 Pulsed Xenon light
 source). We set the light source to single pulses, with an integration
 time of 50msec, a data update range of 5msec, and the scans to average set
 to 20. The spectrophotometer readings were obtained to create a spectral
 curve (OceanView software) from 300-700nm via reflection of the surface of
 the skin. From that curve we were able to calculate brightness (area under
 the curve from 300-700nm), UV chroma (area under the curve from 300-400nm)
 and yellow-orange chroma (area under the curve from 570-620nm). Three
 repeat colour readings were taken from the dorsum, throat, and venter of
 each frog. The probe was disinfected with ethanol after every individual.
 The spectrophotometer was recalibrated using a white diffuse reflection
 standard (OceanOptics WS-1SL) every 30 minutes at a maximum. The spectral
 curve was exported from the OceanView software and imported into R within
 the RStudio interface (RStudio Team 2020, R Core Team 2022). The auc
 function in the MESS package (Ekstrøm 2020) was used to calculate the area
 under the curve for the colour wavelength readings.  We used an
 interpolated average to calculate the mean colour readings at 5nm
 subdivisions (Cassey et al. 2012). This approach ensures that wavelength
 curves are smooth between spectrophotometer readings with slightly
 different sampling slit-widths (Cassey et al. 2012).
   Secondary Sexual
 Characteristics Field trial for colour and calling
 characteristics   Study
 site  Fieldwork was conducted at two sites in the
 Victorian Alpine National Park, Blue Rag Dam (-37.0841881 °S, 147.1217652
 °E, 1600 m elevation) and Dead Timber Creek (-37.019346 °S, 147.239182 °E,
 1590 m elevation). The region is classified as Alpine Sphagnum Bog and
 Associated Fen, which is listed under the Environment Protection and
 Biodiversity Conservation Act (EPBC) 1999 as a nationally threatened
 ecological community. Both sites have a permanent water body and are fed
 by alpine streams and seeps. The vegetation is comprised of snow gum
 (<em>Eucalyptus pauciflora</em>) overstorey with sedges,
 rushes, wetland heaths and sphagnum moss understory and ground cover.
 <em>Batrachochytrium dendrobatidis</em> is endemic to both
 sites. <em> Litoria v. alpina</em> breed in the spring
 following snowmelt and winter torpor (Brannelly et al. 2015,
 2016).    Field
 sampling Our surveys were conducted during the
 austral spring over five sampling times between October 19 and November
 18, 2018. We sampled a total of 45 male <em>L. v. alpina</em>
 for call recordings, colour readings, and infection. 
   We located frogs by triangulating
 their calls and visual spotlighting. Once a calling male was located, we
 approached the animal quietly and carefully to ensure that natural calling
 behaviour was not disturbed. We used a Marantz Professional hand-held
 solid state recorder (PMD6661 MK11, InMusic Japan) and directional
 microphone (Rode Microphones, Sydney, Australia) to record calls of male
 frogs. The recorder was set up one metre away from the calling animal and
 the field team retreated to ten metres away from the frog once the
 recording began to minimise disturbance to the animal. We recorded calls
 using mono recording settings in .wav format at a sampling rate of 48kHz
 with a 24bit resolution. We recorded the calling male for a maximum of 20
 minutes, with the first and last five minutes of the recording disregarded
 to allow the frog to acclimate to our presence. Once the recording was
 complete, we measured the background noise level using a decibel reader
 (specs).     Prior to
 capture, we measured the temperature of the frog and the substrate
 temperature using a dual laser infrared thermometer (RS PRO 8861 ±1°C). We
 captured frogs using clean, gloved hands and by dipnet. We placed each
 frog in individual zip lock bags to prevent potential cross contamination
 of Bd infection. We weighed frogs to the nearest 0.1g with spring scales
 and measured snout-to-vent length (SVL) to the nearest 0.1mm using dial
 callipers.    We collected
 colour readings for male <em>L. v. alpina</em> in the field
 using the same method as outlined in the lab methods above, except in the
 field we only took throat colouration measurements. 
   Call analysis 
 From the 10-minute call recordings, we chose five complete
 calls per male for analysis. We disregarded any calls that were
 overlapping or where the frog was not caught after the recording to
 confirm their infection status. We obtained call and infection data for 39
 male <em>L. v. alpina</em>. We analysed the calls using the
 sound analysis software Raven Pro (Cornell Lab of Ornithology,
 Bioacoustics Research Program). We included the following call
 characteristics in our analysis: call duration (length of the call in
 seconds from the start of the first pulse to the end of the last pulse),
 intercall interval (length of time in seconds between two calls), pulse
 rate (pulses/sec), total number of notes per call, note duration (length
 of note in seconds), internote interval (length of time in seconds between
 two notes), peak note frequency (frequency, Hz, occurring at the highest
 amplitude) and the number of pulses per note. 
   Primary sexual
 characteristics Clinical lab experiment on sperm
 quality and quantity Animals were housed
 individually following the same husbandry procedures as clarified above,
 where animals were housed in individual containers with a gravel and moss
 substrate. Enclosures were flushed with carbon filtered water daily and
 fed twice weekly. In this experiment, we selected individual males (n =
 58) from our <em>L. v. alpina</em> captive colony when they
 were 1 year old and sexually mature in the austral spring. The animals
 were randomly selected from 6 different clutch groups and evenly
 distributed across the exposed and unexposed groups, and randomly
 allocated a number within the two treatment groups (block randomization
 across treatment and clutch of origin). Animals were exposed to the fungal
 pathogen (n = 29) or unexposed control animals (n = 29). We followed the
 same exposure protocol as listed above, however, animals in the infected
 group were exposed to less zoospores (3x105 zoospores in 5mL Milli-Q®
 water). We exposed these animals to a lower infectious dose to try to
 extend the experimental duration to 6 weeks before clinical signs were
 present.    Each week
 animals were swabbed for <em>B. dendrobatidis</em> (following
 the procedures above), weighed and measured. Every two weeks a subset of
 the animals was induced for spermic urine collection. Animals were only
 induced once during the duration of the experiment. We successfully
 collected samples from all 29 control animals (9 on week 2, 10 on week 4
 and 6). Two exposed animals had either cleared infection or were never
 infected at the time of sample collection, therefore the total samples
 collected for the infected animals was 27 individual samples (10 on week
 2, 8 on week 4 and 9 on week 6). Some samples produced few sperm therefore
 not all analyses were conducted on all samples, for example, viability
 analysis was able to be conducted on a total of 48 individuals (see Table
 S3).    We used exogenous
 hormones to stimulate and increase the release of sperm into the spermatic
 urine. We selected the drug Chorulon (a purified hCG) at a dosage of 120
 IU as it has been shown to induce high levels of spermiation in
 <em>L. v. alpina</em> 1 h after exposure (Pham &
 Brannelly, 2022). Prior to administration of hCG we moved the frogs into
 9cm x 9cm x 3cm enclosures containing filtered tap water to allow adequate
 hydration and aid in spermatic urine collection. After 2 h 120 IU of hCG
 was injected into the peritoneal cavity from the ventral side using a 27 g
 needle. We then placed animals back into individual enclosures for 1 h
 before collection of spermatic urine. Prior to sample collection we used a
 Kim wipe to dry cloacal area of the animal. A fire-polished glass Drummond
 microcapillary tube (20μL) was inserted into the cloaca with spermatic
 urine excretion promoted through light massage and rotation of
 microcapillary tube.   To
 determine sperm cell concentration, 2µL spermatic urine was diluted 1:10
 in Simplified Amphibian Ringer solution. Sperm cells were counted at 200x
 using a hemocytometer with 10uL of diluted sample added to each of the two
 chambers. For each sample a minimum of 50 sperm cells were counted
 (repeated for both chambers and averaged) or where 50 cells were not
 present, both chambers were fully counted and every sperm sample was
 included in analysis for analyses of concertation, total sperm cells
 within the sperm produced and volume of sperm. 
   Once sperm cell concentration was
 determined, 30 sec videos were recorded at 200x magnification totalling at
 least 35 sperm cells per sample. If 35 sperm cells could not be recorded
 within the diluted sample, the sample was not included in the analyses.
 Within the videos the sperm cells were scored as non-motile (no observable
 activity), or motile (if any activity was observed over the 30 sec
 observational period). Within the motile category, individual sperm cells
 were categorised as forward moving (the cell moved in a forward motion for
 all or part of the 30 second observation), not-forward moving (the sperm
 head moved but not in a forward motion for all or part of the 30 second
 observation) and stationary moving (the tail moved but the head did not
 for all of part of the 20 second observation). 
   Sperm viability was determined using
 Eosin-Nigrosin staining procedure (Agarwal et al. 2016), with eosin being
 a supravital stain and Nigrosin acting as counterstain increasing contrast
 to better visualise sperm. We added 1µL undiluted spermatic urine sample,
 2µL Eosin Y (1% aqueous) and 2µL Nigrosin (10% aqueous) on a slide and
 mixed with pipette. A thin smear of the stain was created using a clean
 slide. Stained slides were air-dried and then mounted with Permamount
 Mounting Medium (Fisher Scientific). Viability scoring was completed using
 bright-field microscopy at 1000x with oil-immersion objective lens.
 Live/viable sperm cells had white/clear or light pink heads, whereas dead
 sperm cells displayed red or dark-pink heads that were larger in size Fig
 S3. For each sperm sample, a minimum of 90 total sperm cells were
 analyses. If the slide did not contain at least 90 high quality sperm
 cells to analyse, then they were excluded from the analysis.
   Sperm morphology of the live sperm
 cells was conducted via 1000X oil immersion photos taken of individual
 sperm cells stained with Eosin-Nigrosin. Photos were taken and analysed
 using ImageJ software (Wayne Rasband, NIMH) to determine the length of
 head and the length of the tail using the segmented line tool and
 measurement analysis (Pham and Brannelly 2022). We aimed to analyse 20
 individual sperm cells for morphology where possible. If we could not
 analyse at least 10 individual sperm cells per sperm sample, then the
 sample was not included in the analysis. 
   Mating success and offspring
 production Study overview
 The fieldwork for this study was a capture-recapture survey
 where animals were surveyed weekly for six weeks (12 October – 21 November
 2018) at one site in the Victorian Alpine National Park, Blue Rag Dam
 (coordinates withheld because the species is endangered (Lindenmayer and
 Scheele 2017), 1600 m elevation). The study overviewed here also included
 the animals captured within the field study for “Secondary Sexual
 Characteristics” described above.   
 Data collection  Frogs were captured via
 hand with a fresh pair of gloves and individuals were placed in a clean
 ziplock plastic bag. Upon initial capture individuals were sexed and had
 morphometric data including snout-vent length (mm) and mass (g), skin
 swabs for Bd infection load, toe clips, and pictures for identification
 for recaptures taken. Any subsequent captures of an individual included
 all the former listed above minus the acquisition of toe clips. At the
 time of capture, if a male was actively in amplexus with a female, we
 separated the two animals, but noted which female the male was actively
 engaging with. We identified pairs to be in amplexus if both the male had
 a tight grip and the female was not actively attempting to escape the
 male’s grip.    Once the
 animals were collected, photographs were taken using a Panasonic model
 DMC-G5 with a Lumix G VARIO 14-42/F3.5-5.6 lens. Pictures of the left side
 of the groin and dorsal were taken and used for identification of
 individuals. Individuals were swabbed for <em>B. dendrobatidis
 </em>(described above). Toe-tip clips were taken upon initial
 capture of an individual using bleach sterilized scissors, severing one
 toe-tip at the first phalange, and storing the sample in 100%
 ethanol.     Upon each
 survey timepoint, new <em>L. v. alpina</em> egg masses were
 identified, and photos were taken to estimate egg mass size (by estimating
 the proportion of the egg mass in the photograph and counting the visible
 eggs. For each newly laid egg mass, 4 eggs were collected with a
 disposable 2mL pipette from different areas on the outside edge of the egg
 mass (to minimise egg mass disturbance). The individual eggs were stored
 100% ethanol in individual 2mL tubes. 
   In total, 197 unique individuals (179
 males and 18 females) were captured during the field season with 272
 recapture events of males. A total of 451 capture events of male frogs was
 conducted. Female frogs were never recaptured during the sampling period.
 55 egg masses were sampled, and 41 unique egg masses were identified,
 ranging in size from approximately 5 – 220 eggs. 
   Parentage analysis
 To assign parentage to egg masses, all adult toe clips as well
 as three eggs per egg mass (165 total eggs) were genotyped. Tissue samples
 and eggs were sent to Diversity Array’s Technology which utilizes the
 genotype technique DArTseq™. DArTseq™ uses a combination of DArT
 complexity reduction methods and next-generation sequencing platforms
 (Jaccoud et al. 2001, Sansaloni et al. 2011, Kilian et al. 2012, Cruz et
 al. 2013).     A dataset of
 approximately 85,374 SNPs was returned and these were stringently filtered
 in R using the RStudio interface (RStudio Team 2020, R Core Team 2022)
 using the dartR package (Gruber et al. 2018) to 3570 SNP loci that had a
 call rate of higher than 90% and reproducibility higher than 98%. From
 these SNPs and the successfully genotyped individuals we used the program
 Colony (Wang 2004, 2012, 2013a, 2013b, Jones and Wang 2010a, 2010b) to
 determine parentage of egg masses.   
 Determination of male breeding status
 Within our genetic parental analysis, we found that no egg
 masses were produced between males and females that we captured in
 amplexus with each other. While amplexus does not always lead to spawning
 (Jennions et al. 1992, Orton et al. 2023, Brannelly et al. 2023), in many
 cases the females in amplexus pairs began to oviposit directly after
 capture. We believe that we disturbed the breeding pairs for sample
 collection, and while no breeding event took place, it would have if we
 had not interrupted. Therefore, when we determined the breeding status of
 the males at each sampling point throughout the breeding season, we
 included both egg masses produced and observed pairs in amplexus.
 Additionally, by including observations of both amplexus and the
 production of egg masses we bolstered our results: we associated 21 egg
 masses to 20 male capture events throughout the breeding season, and 18
 instances of amplexus.
description: # Devastating disease can cause increased breeding effort and success that
 improves population resilience
 [https://doi.org/10.5061/dryad.vx0k6dk0f](https://doi.org/10.5061/dryad.vx0k6dk0f) ## Description of files The files included in this data set were collected from the field and from laboratory infection experiments. #### Field data: * `Lva_Field_Breeding.csv` indicates the males that were captured during the capture mark recapture study, and data were collected on the individual males. Species is Lva for all, which is Litoria verreauxii alpina, and the site is BR for all, which means they were all collected at the same site. Date is in day_mo_yyyy format, and Julian_day is also provided. Week indicates the week of the CMR trial. Each animal was given a day of `Field_ID`, and if they were recaptured, the individual the `Recapture_ID` corresponds to the `Concatenated_Field_ID` on first capture. `Individual_ID` is the unique individual identification used in analyses. `Recapture` column is 0 = not recaptured and 1 = recaptured. `SVL` is in mm, `Mass` is in g. `Sex` is M (male) for all animals. `Call_at_capture` is whether the animal was calling at the point of capture (1 = yes, 0 = no). `Call_Bag` is whether the animal was called while captured at any point during processing. `Amplexus` is whether the animal was found in amplexus with a female. `Repro_effort` is a 1 if `Call_at_capture`, `Call_Bag` and/or `Amplexus` was a 1, and 0 if all three other columns were 0. qPCR data for Bd infection dynamics is included where `copies.swab` is the qPCR results indicated DNA copies of Bd per sample, and `Infection_Load` is log transformed `copies.swab` and the cell is NA if no infection was detected. This column was used in the analysis. `Infection_Status` is whether the animal was positive (1) or negative (0) for that swab sample. `Bd_status` is `Infection_Status` but in words, not numbers. The `breeding_event_status` is whether the male spawned an egg clutch within the proceeding week of capture or if they were found in amplexus with a female at the point of capture. Environmental and site data are the rest of the variables, where `Call.Survey` is an estimate of the number of animals calling at the start of the survey within a 3-minute window. `Start_Time` and `End_Time` are the start and end times of the survey on that day. Water quality measurements are `pH`, `Water_Temp` (degrees C), `Conductivity` (µS), `TDS` (total dissolved solids, ppm). Weather data includes `Air_Temp` (degrees C), `Wind_Speed` (m/s), and `Site_Notes` are observed weather notes. The last section of this data frame includes the number of egg masses assigned to each individual frog on the collection day. `Number_eggmasses` is the number of egg masses attributed to each male frog. `Status_eggmass` is whether the male sired an egg mass at that point in time (0 = no, 1 = yes). The `breeding_event_status` is a 1 (displayed breeding effort) if `Status_eggmass` and/or `Amplexus` is a 1, and 0 (no breeding effort displayed) if both `Status_eggmass` and `Amplexus` were 0 for that timepoint. * `Lva_Field_Calling.csv` is the animals for which we were able to collect calling recordings from. The metadata and field data columns are the same as in the previous dataframe. Additional columns are: `Lva_Calling`, which is 1 if Litoria verreauxii alpina were heard calling during the call survey, and 0 if not heard calling. `Crsi_Calling` is 1 if Crinia signifera was heard calling during the call survey, and 0 if not heard calling. Calling data about the actual recording: The recorder indicates which call recorder is used (1 or 2). `Recording_date` is in the format of the call recording file. `Recording_ID` is the call recording number within the recording file name. `Call_rate` is the number of calls within the recording. `Call_no.` is the call number within the recording. `Call_duration_sec` is the length of the call in seconds from the start of the first pulse to the end of the last pulse. `Intercall_sec` is the length of time in seconds between two calls. `Notes_total` is the number of notes per call, `Note_no.` is the note number within the call, `Note_duration` is the length of the note in seconds, `Internote` is the internote interval, which is the length of time in seconds between two notes. `Pulse_rate` is pulses/sec within the note, `Peak_note_freq` is the frequency, Hz, occurring at the highest amplitude. `Pulse_per_second` is the number of pulses per second within the note. `ID_call` and `ID_call_note` are the labels given to each call and note recorded. `Temp.of.Animal` is the frog temperature (degrees C) directly before capture. `Temp.of.Substrate` is the temperature of the substrate on which the frog was captured (degrees C). `Substrate` is what the frog was sitting on before capture. `Decibel.Max` is the max decibel reading (in decibels), and `Decibel.min` is the minimum decibel reading during the call recording period. * `Lva_Field_Colour.csv` are animals for which we were able to collect colour data. The metadata and field data columns are the same as in the previous dataframes. Each line of data is a colour analysis from one spectrophotometer reading (`Frog_reading`), and three readings were taken from the throat of each frog. Colour reading data is the area under the curve from the spectrophotometer output, `brightness` (area under the curve from 300-700nm), `UVchroma` (area under the curve from 300-400nm) and `yellow_orange` chroma (area under the curve from 570-620nm). #### Laboratory infection experiment data * `Lva_InfectionExperiment_Colour.csv` contains animals from one infection experiment where we monitored the colouration of animals throughout an experimental infection. `Frog_ID` is the individual frog that was repeatedly measured throughout the experiment. `Clutch` is the egg clutch that the individual came from. `Date` is the date of the sample, and `Week` is the week of the experiment. `Week` 0 is on the day of inoculation. `Tank` is the number of `Frog_ID`. `Treatment` is whether the animal was an unexposed control or exposed to the fungal pathogen. `Room.temp` is the temperature of the room on the day in degrees C. `Humidity` is the humidity percent in the room on the day. `SVL_mm` is in mm, `Mass_g` is in g. Arm1, 2, and 3 are repeated forearm widths in mm, and Thumb1, 2, and 3 are repeated nuptial pad lengths in mm. Infection columns are the same as data frames described above. `Frog_reading` is the location of the skin area measured (dorsum, venter and throat). Each line of data is one colour analysis from one spectrophotometer reading, and three readings were taken from each Frogreading of each frog. The colour readings are the same as `Lva_Field_Colour.csv`. #### Sperm quality and quantity analyses: * `Lva_Sperm_Concentration.csv` contains animals from one infection experiment where we monitored sperm parameters of animals throughout an experimental infection. Each frog was assigned to a `Group` (infected/unexposed), and sperm was taken only once per frog. `Mass_Date` is the date that the sperm sample was taken. `Week` 0 is on the day of inoculation. Column titles are consistent with the dataframes above. `Tube_length` is the length of the capillary tube used for collection in mm. `Tube_volume` is the total volume of the capillary tube used for collection in uL. `Urine_length` is the length of the urine within the capillary tube. `Urine_volume` is in uL and is calculated from the total `Tube_volume` and `Urine_length`. `UrineVolumeUsed` is the μL of spermic urine used on the haemocytometer. `SAR.volume` is the volume of Simplified amphibian ringers solution used to dilute the spermic urine. The `dilution` is the dilution factor of spermic urine to SAR. Count_1 to 4 is the haemocytometer well counts. Hem_cells_counted_1 to 4 are the number of haemocytometer cells counted for each Count_#. Together with `dilution`, they were used to calculate `Total_conc`, which is the sperm concentration (cells/mL) of sperm within the spermic urine. `Total_sperm` is the total number of sperm, using the concentration and volume to calculate the total. * `Lva_Sperm_Motility.csv` uses the same metadata as in the previous dataset. The number of `non-motile` sperm is `non_motile`. `M_forward` is sperm with forward progression during the 30-second sperm video. `M_moving` is sperm moving but not in a forward progression, and `M-stationary` is some movement but the head does not move from its spot in the 30-second video. `no.video` is the number of videos that were analysed and counted. `M_MandF` is M_moving + M_forward. `total_sperm_measured` is the sum of M_moving + M_forward + M_stationary + non_motile. `Prop_motile` is the proportion of moving to the total number of sperm analysed, and this was what was used in analyses. * `Lva_Sperm_Viability.csv` uses the same metadata as in the previous datasets. `Magnification` is the microscope magnification on which the sperm were assessed. `Dead_pinkred` is the number of dead sperm, `Alive_clearpink` is the number of alive sperm. `Total_sperm_counted` is the total number of sperm counted (Dead_pinkred + Alive_clearpink). `Proportion_alive` is used for analyses, and this is Alive_clearpink/Total_sperm. * `Lva_Sperm_Morphology.csv` uses the same metadata as in the previous datasets. 20 sperm were measured per individual sample. `Photo_ID` is the name of the photo for the data collection. `Sperm_number` is the number of sperm measured. `head_um` and `Head_length_pxl` are the length of the sperm head in µm and pxl. The `tail.um` and `Tail_lenght_pxl` are the length of the tail in µm and pxl.