DOI: 10.5061/dryad.rv15dv48t
title: Reproductive allocation in Nicrophorus maliger and Nicrophorus guttula

description: Abstract: The cost of reproduction hypothesis suggests that allocation to
 current reproduction constrains future reproduction. How organisms accrue
 reproductive costs and allocate energy across their lifetime may differ
 among species adapted to different resource types. We test this by
 comparing lifetime reproductive output, patterns of reproductive
 allocation, and senescence between two species of burying beetles,
 Nicrophorus marginatus and N. guttula, that differ in body size, across a
 range of carcass sizes. These two species of burying beetles maximized
 lifetime reproductive output on somewhat different–sized resources. The
 larger N. marginatus did better on large and medium carcasses while the
 smaller N. guttula did best on small and medium carcasses. For both
 species, reproduction is costly and reproduction on larger carcasses
 reduced lifespan more than reproduction on smaller carcasses. Carcass size
 also affected lifetime reproductive strategies. Each species’ parental
 investment patterns were consistent with terminal investment on carcasses
 on which they performed best (optimal carcass sizes). However, they
 exhibited reproductive restraint on carcass sizes on which they did not
 perform as well. Reproductive senescence occurred largely in response to
 carcass size. For both species, reproduction on larger carcasses resulted
 in more rapid senescence. These data suggest that whether organisms
 exhibit terminal investment or reproductive restraint may depend on type
 and amount of resources for reproduction.
description: 2.1. Burying Beetle Natural History Burying
 beetles locate small vertebrate carcasses and use them as food resources
 for themselves and their offspring. Males and females compete with
 individuals of the same sex until a single pair, typically those with the
 largest body sizes, dominate the carcass [37,38]. Similar to within
 species competitive dynamics, larger species will displace smaller species
 when they co–occur on a carcass [10,31]. The winning pair of beetles
 buries the carcass under the soil, removes the feathers or hair, shapes
 the carcass into a ball, and coats it with oral and anal secretions that
 help prevent microbial growth. During carcass preparation, the female lays
 eggs in the soil, and larvae usually hatch on or after the fourth day.
 Parents adjust brood size through filial cannibalism, regurgitate food to
 newly hatched larvae, and provide defense of the carcass and larvae from
 conspecifics. Parental care continues until larvae disperse into the soil
 to pupate [31]. 2.2. Experimental Design
 We collected both <i>N. marginatus </i>and<i>
 N. guttula</i> at Goshen Ponds (39° 57.476’N, 111° 51.426’W) and
 Utah Lake Wetland Preserve (40° 6.933’N, 111° 47.589'W) in central
 Utah during June 2011 and July 2012 using pitfall traps baited with aged
 chicken. We transported beetles back to Brigham Young University and
 established laboratory populations for each species by breeding
 wild–caught pairs on a 30 g carcass. We kept newly eclosed offspring in
 small plastic containers (11.3 cm L x 7.6 cm W x 5.7 cm H), provided them
 ad libidum raw chicken liver, and maintained them on a 14L:10D cycle (a
 natural photoperiod for the summer breeding season at the source
 location). Beetles used in this experiment were F1, F2, and F3
 individuals, and all crosses used different family lines to ensure no
 inbreeding occurred. At 28 days (± 1 day) from
 eclosion, we randomly assigned females from both species to one of six
 carcass size treatments and one nonreproductive treatment (12 replicates
 for each treatment for a total of 84 females of each species). In six of
 the treatments, we allowed females to reproduce throughout their lifetime
 on one of six carcass sizes (5 g, 10 g, 20 g, 30 g, 40 g, or 50 g, ±1.0 g
 lab mouse carcasses; this range in size covers the range of carcass sizes
 available in the natural environment). For each reproductive bout, we
 placed each female with a virgin male (at least 21 days old, to ensure
 sexual maturity) on a carcass of their assigned size in a plastic
 container (20.3 cm L x 15.2 cm W x 9.8 cm H) filled with approximately 4.5
 cm (depth) of commercially purchased topsoil and allowed them to
 reproduce. After 48 h, we removed males from the carcass to isolate the
 reproductive investment patterns of females (males were present only
 during the beginning of the carcass preparation phase). At the end of each
 reproductive attempt (defined as the point when all larvae dispersed from
 the carcass into the soil), we removed the females, placed them each in a
 small, individual container, and provided them with a moistened paper
 towel for water and chicken liver ad libidum. After 48 h, we placed
 females on a new carcass (of the same size as their previous reproductive
 attempt) with a virgin male and allowed them to produce offspring. This
 cycle was repeated until the female died. To assess effects of
 reproduction on lifespan, our seventh treatment was a “non–reproducing”
 treatment where females were fed on chicken liver (0.5 g to 1 g twice per
 week), but were not allowed to breed throughout their life.
 For each treatment, we weighed females and measured their
 pronotum width at 28 days of age (±1 day), and when the female died we
 recorded her lifespan. For the six reproducing treatments, we weighed
 females before and after each reproductive attempt. We monitored each
 female and her brood daily to determine brood size and timing of larval
 dispersal. If, after 7 days, no offspring had appeared on the carcass, we
 designated the brood as a failure, and removed the female, gave her food,
 and isolated her for 48 h, then allowed her to breed again on a fresh
 carcass with a new virgin male. We recorded the initial and final number
 of offspring and mass of offspring as they dispersed into the soil for
 each reproductive attempt. 2.3. Statistical
 Analyses 2.3.1. Analysis of Optimal Carcass
 Size Our first goal in the experiment was to determine
 optimal carcass sizes for each species. We defined “optimal” as the
 carcass size that resulted in the greatest number of offspring produced
 over a lifetime. To determine optimal carcass size, we used a general
 linear model to examine the effects of carcass size and species on
 lifetime number of offspring (GLM procedure; SAS 9.3 SAS Institute, Cary,
 NC, USA). In the model, carcass size and species were main effects, and we
 included an interaction between carcass size and species, and standardized
 female body size (pronotum width) as a covariate. As noted above, body
 size varies between the two species. We were interested in effects of body
 size within each species, and we did not want to confound differences in
 body size between species with within species variation, so we
 standardized body size across species by creating a z–score centered on
 the mean of each species. The response variable, lifetime total number of
 offspring, was log transformed to meet assumptions for the parametric
 model. One <i>N. guttula </i>female from the 20 g carcass size
 never reproduced and was removed from all analyses.
 Total mass of offspring over the lifetime (i.e., total number of
 offspring multiplied by mean offspring mass per brood) is sometimes used
 to represent evolutionary fitness. We analyzed patterns of total mass of
 offspring over a lifetime using the same model as that used for total
 number of offspring. Results were consistent with results obtained from
 total number of offspring, so we present only total number of offspring in
 this paper. 2.3.2. Analysis of Patterns of Reproductive
 Allocation Our second goal was to determine if
 reproductive allocation and resulting senescence followed a pattern of
 terminal investment or reproductive restraint. We used four response
 variables to characterize contrasting patterns of reproductive allocation
 as follows: lifespan, lifetime number of reproductive bouts, mass change
 of females through time, and proportion of offspring culled through time.
 Terminal investment would be characterized by shorter lifespans, fewer
 reproductive bouts, negative or neutral mass gain, and fewer offspring
 culled at older ages. In contrast, reproductive restraint would be
 characterized by longer lifespans, more reproductive bouts, positive mass
 gain, and more offspring culled at older ages. To test
 for differences in lifespan, we used a general linear model (GLM
 procedure; SAS 9.3 SAS Institute, Cary, NC, USA) and for differences in
 number of reproductive bouts we used a generalized linear model (GenMod
 procedure; SAS 9.3 SAS Institute, Cary, NC, USA). For each model, carcass
 size and species were main effects, and we included an interaction between
 carcass size and species, and standardized female body size (pronotum
 width) as a covariate. As noted above, body size varies between the two
 species. We were interested in effects of body size within each species
 and we did not want to confound differences in body size between species
 with within species variation, so we standardized body size across species
 by creating a z–score centered on the mean of each species. The
 non–reproducing treatment was only included in the model for lifespan.
 Data for lifespan met the assumptions of the parametric model and was not
 transformed. For the number of successful reproductive bouts, we assumed a
 Poisson distribution and used a log–link function. To
 test for differences in mass change and proportion brood culled within
 lifetimes, we used a generalized linear mixed model (GLMM; GLIMMIX
 procedure; SAS 9.3 SAS Institute, Cary, NC, USA). Mass change was measured
 as mass of female at the end of the reproductive bout minus mass of the
 female at the beginning of the reproductive bout. Mass loss would be
 observed as a negative number and mass gain would be positive. Mass change
 is a continuous variable, and raw data met assumptions for a parametric
 model, so no transformations were used. Proportion brood culled was
 measured as number of offspring culled relative to initial brood size, so
 we assumed a binomial distribution with a logit–link function. We used a
 repeated measures design to analyze patterns of allocation through time.
 Species, carcass size, and age (indexed by reproductive bout) were
 predictor variables (i.e., main effects). We used standardized female body
 size as a covariate and included all two–way and three–way interactions
 among main effects. A single <i>N. guttula</i> female never
 reproduced and thus we removed her from all analyses. Because we had
 multiple measures of the same individual through time, individual ID was
 used as a random effect in the model. 2.3.3. Analysis
 of Patterns of Reproductive Senescence To assess
 patterns of reproductive senescence, we used three response variables as
 follows: initial brood size, final brood size, and offspring body mass at
 dispersal. An increased rate of senescence would be characterized by a
 negative slope of initial offspring number and final offspring number with
 increasing age; whereas, a decreased rate of senescence or delayed
 senescence would be characterized by a zero slope of initial offspring
 number and final offspring number with increasing age. Because both the
 female and the offspring feed exclusively on the carcass during brood
 development, fewer offspring should result in larger offspring body size.
 Thus, senescence would result in fewer but larger offspring in older
 individuals; whereas, delayed senescence would result in about the same
 number and size of offspring with increasing age. To
 test for differences in initial brood size, final brood size, and
 individual offspring mass within lifetimes we used generalized linear
 mixed models (GLMM; GLIMMIX procedure; SAS 9.3 SAS Institute, Cary, NC,
 USA). We ran separate models for each of the three response variables.
 Initial brood size was the number of larvae that first appeared on the
 carcass before culling had occurred. Final brood size was the number of
 larvae that dispersed into the soil and represents the brood size after
 culling has occurred. For each trait we used a repeated measures design to
 analyze patterns of senescence through time. Species, carcass size, and
 reproductive bout (age) were predictor variables (i.e., main effects). We
 used standardized female body size as a covariate and included all two–way
 and three–way interactions. For both initial and final brood sizes, we
 assumed a Poisson distribution and used a log–link function. Offspring
 mass was a continuous variable, so we used a log–link function. Because we
 had multiple measures of the same individual through time, individual ID
 was used as a random effect in the model. A single <i>N.
 guttula</i> female never reproduced and thus we removed her from all
 analyses. In addition, we removed two bouts from a single female from the
 analysis for mean offspring mass because in each bout, only two offspring
 were produced and they were abnormally small (1/5 the size of any other
 offspring).
description: Two datasets: first is the
 "lifetime" dataset, "." is missing data
 Variables (columns) are as follows: species
 (1=N. marginatus 2=N. guttula) ID - individual ID
 number carcass size (0=5g, 1=10g, 2=20g, 3=30g, 4=40g,
 5=50g, and 7= non-reproductive) lifespan (in
 days) size (pronotum width of female)
 sbouts = successful bouts  tbouts = total
 bouts totoffmass = total offspring mass
 numoffspring = number of offspring  
 Second data set is "within", "." is missing
 data Variables (columns) are as follows:
 species (1=N. marginatus 2=N. guttula) carcass
 size (0=5g, 1=10g, 2=20g, 3=30g, 4=40g, 5=50g) ID -
 individual ID number age - reproductive bout
 cmass - actual carcass mass in g pw - pronotum
 width  startmass - mass of female at beginning of
 reproductive bout endmass - mass of female at end of
 reproductive bout startbrood - initial number of
 offspring in brood  finalbrood - final number of
 offspring in brood  moffsize - mean offspring
 size totoffmass - total offspring mass per
 brood masschange - mass change of female during
 bout efficiency - brood mass divided by carcass
 mass