Alternative Concepts of Reproductive Effort, Costs of Reproduction, and Selection in Life-History Evolution

An outline for an organismic theory of reproductive tacticsis presented to develop the demographic theory of optimal reproductivetactics into a more realistic theory of life-history evolution.Reproductive effort—denned as the proportion of resourcesinvested in reproduction—and the costs in somatic investmentdo not automatically result in survival costs. Both the conditionswhere survival costs are produced and the conditions where reproductioncan take place without survival costs are specified. Compensationand threshold hypotheses are put forward to allow weaker correlationsbetween reproduction and survival than the trade-off hypothesis,which assumes direct impacts by reproductive effort on survival.Furthermore, reproductive tactics are unlikely to be mouldedby the demographic forces of selection only. An empirical exampleis shown where residual reproductive value played no significantrole in the evolution of reproductive tactics. Selection probablyoperates not on separate life-history traits but on whole organismsthrough their entire life-history. The structural and physiologicalintercouplings between separate traits can result in phenotypicopportunity sets where selection can mould life-history traitsonly within the constraints of the opportunity sets. Optimizationtheory has provided an efficient technique for modelling andmaking predictions. However, organismic selection does not necessarilyoptimize adaptive strategies but eliminates unfit strategies.Life-history theory, and evolutionary theory in general, canbe developed along alternative logical lines when differenthypotheses are generated on how selection operates.     

Genetic Variation in Reproductive Investment Across an Ephemerality Gradient in Daphnia pulex

Species across the tree of life can switch between asexual and sexual reproduction. In facultatively sexual species, the ability to switch between reproductive modes is often environmentally dependent and subject to local adaptation. However, the ecological and evolutionary factors that influence the maintenance and turnover of polymorphism associated with facultative sex remain unclear. We studied the ecological and evolutionary dynamics of reproductive investment in the facultatively sexual model species, Daphnia pulex. We found that patterns of clonal diversity, but not genetic diversity varied among ponds consistent with the predicted relationship between ephemerality and clonal structure. Reconstruction of a multi-year pedigree demonstrated the coexistence of clones that differ in their investment into male production. Mapping of quantitative variation in male production using lab-generated and field-collected individuals identified multiple putative quantitative trait loci (QTL) underlying this trait, and we identified a plausible candidate gene. The evolutionary history of these QTL suggests that they are relatively young, and male limitation in this system is a rapidly evolving trait. Our work highlights the dynamic nature of the genetic structure and composition of facultative sex across space and time and suggests that quantitative genetic variation in reproductive strategy can undergo rapid evolutionary turnover.     

Intraspecific Variation of Reproductive Traits in a Mediterranean Lizard: Clutch, Population, and Lineage Effects

Widely distributed terrestrial ectotherms from the southern European peninsulas show patterns of subdivision (related to isolation in temperate refugia) that allow us to test the relative importance of phylogeographic lineage, population of origin and familial effects as sources of variation for life-history traits. We collected gravid females from 15 geographically separated populations of the lacertid lizard Psammodromus algirus, a widely distributed species with well differentiated eastern and western lineages. We incubated eggs under two treatments of constant (28°C) and fluctuating (28 ± 4°C) temperature, and we examined clutch, population, and lineage effects on several traits of females, eggs, and hatchlings. Incubation time was mainly explained by differences between lineages, but it was also influenced by population and female effects. Within each lineage, incubation was shorter at cooler and wetter sites, and for a given climate it was shorter for eastern than for western populations, suggesting that countergradient variation has evolved independently in the two lineages. Female size, clutch size, and relative fecundity were primarily influenced by inter-population differences, a pattern that seemed attributable to environmental differences in productivity, because mean female size was positively correlated with a gradient of increasing precipitation and decreasing temperature. Clutch size was mainly, but not entirely, dependent on female SVL, suggesting both a proximate effect of local conditions and intrinsic differences among populations. Females from drier and warmer sites produced larger hatchlings. Mean egg mass was mainly determined by familial effects. Eggs incubated at a constant temperature hatched earlier than did their siblings incubated at fluctuating temperatures, a fact that could be explained by considering that in Mediterranean environments developmental rate might increase at a lower speed above average incubation temperature than it does decrease below it.     

Costs and Benefits of Competitive Traits in Females: Aggression,Maternal Care and Reproductive Success

Recent research has shown that female expression of competitive traits can be advantageous, providing greater access to limited reproductive resources. In males increased competitive trait expression often comes at a cost, e.g. trading off with parental effort. However, it is currently unclear whether, and to what extent, females also face such tradeoffs, whether the costs associated with that tradeoff overwhelm the potential benefits of resource acquisition, and how environmental factors might alter those relationships. To address this gap, we examine the relationships between aggression, maternal effort, offspring quality and reproductive success in a common songbird, the dark-eyed junco (Junco hyemalis), over two breeding seasons. We found that compared to less aggressive females, more aggressive females spent less time brooding nestlings, but fed nestlings more frequently. In the year with better breeding conditions, more aggressive females produced smaller eggs and lighter hatchlings, but in the year with poorer breeding conditions they produced larger eggs and achieved greater nest success. There was no relationship between aggression and nestling mass after hatch day in either year. These findings suggest that though females appear to tradeoff competitive ability with some forms of maternal care, the costs may be less than previously thought. Further, the observed year effects suggest that costs and benefits vary according to environmental variables, which may help to account for variation in the level of trait expression.     

Occurrence,Plasticity, and Evolution of the vpma Gene Family,a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.Bacteria of the Mycoplasma genus belong to the class Mollicutes and represent a remarkable group of organisms that derived from the Firmicutes lineage by massive genome reduction (41, 51). Consequent to this regressive evolution, modern mycoplasmas have been left with small genomes (580 to 1,400 kb), a limited number of metabolic pathways, and no cell wall. Due to these particularities, members of the Mycoplasma genus have often been portrayed as “minimal self-replicating organisms.” Despite this apparent simplicity, a large number of mycoplasma species are successful pathogens of humans and a wide range of animals, in which they are known to cause diseases that are often chronic and debilitating (1, 33). The surface of their single membrane is considered a key interface in mediating adaptation and survival in the context of a complex, immunocompetent host (10, 13, 34, 40). Indeed, mycoplasmas possess a highly versatile surface architecture due to a number of sophisticated genetic systems that promote intraclonal variation in the expression and structure of abundant surface lipoproteins (9, 50). Usually, these systems combine a set of contingency genes with a molecular switch for turning expression on or off that is based on either (i) spontaneous mutation (slipped-strand mispairing), (ii) gene conversion, or (iii) specific DNA rearrangements (9). While high-frequency phenotypic variation using the two first mechanisms has been described thoroughly for other bacteria (47), switching of surface components by shuffling of silent genes at a particular single expression locus has been studied mainly in mycoplasmas (3, 8, 14, 16, 23, 39, 43).Mycoplasma agalactiae, an important pathogen responsible for contagious agalactia in small ruminants (listed by the World Organisation for Animal Health), possesses a family of lipoproteins encoded by the vpma genes for which phase variation in expression is driven by a “cut-and-paste” mechanism involving a tyrosine site-specific recombinase designated Xer1 (16). Data previously gathered with the PG2 type strain identified a single vpma cluster (42) composed of six vpma genes adjacent to one xer1 gene (Fig. ​(Fig.1A).1A). Based on fine genetic analyses, Xer1 was further shown to mediate frequent site-specific DNA rearrangements by targeting short DNA sequences located upstream of each vpma gene (8, 16). While some vpma rearrangements can be phenotypically silent, others result in Vpma on-off switching by linking a silent vpma gene sequence immediately downstream of the unique vpma promoter. Because site-specific recombination can be reciprocal, the initial vpma configuration can be restored without a loss of genetic information.Open in a separate windowFIG. 1.Comparison of M. agalactiae vpma loci between the PG2 type strain and strain 5632. Schematics represent the organization of the vpma loci in clonal variant 55.5 derived from PG2 (16, 42) (A) and in clonal variant c1 derived from strain 5632 (B). (C) Counterpart of locus II₅₆₃₂ in PG2 showing the absence of vpma genes in this region. (D) The presence of two distinct loci in 5632 was confirmed by PCR, using the primer pair xerF-phydR or xerF-agpR, and the resulting amplicons are shown. The locations of the primers are indicated by arrowheads in panels A, B, and C. Large white arrows labeled with letters represent Vpma CDSs. The positions of the promoters are represented by black arrowheads labeled “P.” The two non-Vpma-related CDSs (abiG_I and abiG_II) are indicated by large arrows filled with a dotted pattern. ISMag1 elements are indicated by hatched boxes. Recombination sites downstream of each vpma gene are indicated by black dots. An asterisk indicates that the corresponding vpma gene is present at two distinct loci. Schematics were drawn approximately to scale. HP, hypothetical protein; CHP, conserved hypothetical protein. Small letters and bars indicate the positions of short particular sequences mentioned in the text and in Fig. ​Fig.33 and ​and4.4. The pictures on the left side of panels A and B illustrate the variable surface expression of Vpma, as previously described (8, 17). These correspond to colony immunoblots using Vpma-specific polyclonal antibodies recognizing PG2 VpmaW (α W) and VpmaY (α Y) epitopes.The vsa family of the murine pathogen M. pulmonis (3, 39), the vsp family of the bovine pathogen M. bovis (2, 24), and the vpma family of M. agalactiae all generate intraclonal surface diversity by using very similar molecular switches (23), although their overall coding sequences seem to be specific to the Mycoplasma species. DNA rearrangements also govern phase variation of the 38 mpl genes of the human pathogen M. penetrans (27, 35, 38). However, in this mycoplasma species the molecular switch is slightly different, since each mpl gene possesses its own invertible promoter (19). In M. penetrans, the individual expression of each mpl gene can then be switched on and off in a combinatory manner, resulting in a large number of possible Mpl surface configurations. Since M. pulmonis, M. bovis, and M. agalactiae all belong to the Mycoplasma hominis phylogenetic cluster (48) and are relatively closely related, while M. penetrans belongs to the distinct Mycoplasma pneumoniae phylogenetic cluster (30, 48), it is tempting to speculate that the vsa, vsp, and vpma systems were all inherited from a common ancestor and that the bulk of their coding sequences evolved independently in their respective hosts while the molecular switch mechanism was retained.In so-called “minimal” bacteria, the occurrence of relatively large genomic portions dedicated to multigene families, with genes encoding phase-variable, related surface proteins, suggests that they serve an important function(s). Data accumulated over the years for several mycoplasma species tend to indicate that one general purpose of these systems is to provide the mycoplasma with a variable shield that modulates surface accessibility in order to escape the host response and to adapt to rapidly changing environments (10, 11, 13, 40, 50). On the other hand, the sequences of phase-variable proteins are relatively conserved within one species but divergent between species, suggesting a more specific role for these molecules.The role of the Vpma family of M. agalactiae has yet to be elucidated, but it was recently shown that Vpma switches in expression occur at a remarkably high rate in vitro (10⁻² to 10⁻³ per cell per generation) (8, 17). The vpma systems described for PG2 (16) and another M. agalactiae strain, isolated in Israel (in which Vpmas were designated Avg proteins [14]), both revealed a repertoire of six vpma genes and only one promoter, suggesting that in M. agalactiae the number of Vpma configurations is limited to six. This contrasts with the situation commonly found in other Mycoplasma variable systems, which can offer a larger mosaic of surface architecture because of the concomitant switches of several related surface proteins and/or because of a larger number of phase-variable genes.To further understand the degree of diversity that can be generated at the surface of M. agalactiae, we analyzed the vpma gene content of a field strain, 5632, whose genome was recently sequenced by our group (unpublished data). The present study shows that 5632 contains a total of 23 vpma genes distributed in two distinct loci that both contain a recombinase gene. Further genomic and proteomic analyses indicated that the capacity of 5632 to vary its Vpma surface architecture is far more complex than that described for the type strain. Unlike the case for PG2, both 5632 vpma loci are associated with several mobile genetic insertion elements (IS) that could play an evolutionary role in the dynamics of vpma repertoires, as suggested by data presented here. One 5632 vpma locus contains open reading frames (ORFs) that are highly conserved in both M. bovis, a closely related bovine mycoplasma, and the phylogenetically distant mycoplasmas of the M. mycoides cluster, which are also important ruminant pathogens. Whether these were acquired through evolution or through horizontal transfer is discussed. The present study reveals an additional degree of complexity for the Vpma system and further suggests that some field strains might have more dynamic genomes and a more variable surface than was first estimated (42).     

Reproductive Trade-Offs and Direct Costs for Males in Arthropods

Until 30 years ago, the emphasis on reproductive costs for males was mainly on costs related to mate searching, courtship and fighting with rival males. However, costs for males are substantial and varied and often resemble the more thoroughly studied female reproductive costs. Costs can be referred to as trade-off costs, where investment in reproductive activity comes at the expense of another important activity or fitness component. Investment in reproduction at the expense of longevity and future reproduction is the ultimate cost, because it affects fitness directly. In contrast, flawed performance (e.g., of the immune system) is perceived as a mechanistic trade-off, because it affects fitness indirectly through a mediator (i.e., parasites). Finally, direct costs refer to direct measurements of the energy expenditure during involvement in reproduction-related activities. Both direct and mechanistic trade-off costs often result in decreased longevity compared to unmated males (an ultimate cost). Males incur costs during different reproductive phases: before copulation, when producing sperm, while searching for, courting and copulating with females, and subsequently when guarding females or taking care of offspring. This synthesis follows previous pioneering reviews addressing specific aspects of male costs, but strives to summarize all known male reproductive cost types more comprehensively, including their classification. We suggest several directions for targeted future research. While costs for males have been fairly well described, it is now necessary to uncover the ecological and evolutionary factors responsible for differences between closely related species and systems and to better link between directly-measured costs, mechanistic trade-off costs and ultimate trade-off costs.     

务川黑牛mtDNA遗传变异及起源进化分析

目的:测定务川黑牛mtDNA D-loop区全序列,以了解其遗传背景。方法:采用PCR直接测序方法测定务川黑牛mtD-NA D-loop区全序列。结果:32个个体D-loop区的全序列中,四种碱基A、T、C和G含量分别为:33.1%、28.4%、25.0%和13.5%,A+T平均含量61.5%。经过序列比对,共检测到57个变异位点,占所测mtDNA序列总长的6.26%,其中转换54个,颠换3个;产生17种单倍型,其中6种是普通牛单倍型,11种是瘤牛单倍型;平均核苷酸差异为22.623,单倍型多样度0.901±0.039,核苷酸多样度0.0249±0.00147。结论:务川黑牛这一品种遗传多样性非常丰富,据此构建的NJ进化树显示务川黑牛含有普通牛和瘤牛的血统。     

Genetic Variation at the BDNF Locus: Evidence for Association with Long-Term Outcome after Ischemic Stroke

Background and Purpose

Rates and extent of recovery after stroke vary considerably between individuals and genetic factors are thought to contribute to post-stroke outcome. Brain-derived neurotrophic factor (BDNF) plays important roles in brain plasticity and repair and has been shown to be involved in stroke severity, recovery, and outcome in animal models. Few clinical studies on BDNF genotypes in relation to ischemic stroke have been performed. The aims of the present study are therefore to investigate whether genetic variation at the BDNF locus is associated with initial stroke severity, recovery and/or short-term and long-term functional outcome after ischemic stroke.

Methods

Four BDNF tagSNPs were analyzed in the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS; 600 patients and 600 controls, all aged 18–70 years). Stroke severity was assessed using the NIH Stroke Scale (NIHSS). Stroke recovery was defined as the change in NIHSS over a 3-month period. Short- and long-term functional outcome post-stroke was assessed using the modified Rankin Scale at 3 months and at 2 and 7 years after stroke, respectively.

Results

No SNP was associated with stroke severity or recovery at 3 months and no SNP had an impact on short-term outcome. However, rs11030119 was independently associated with poor functional outcome 7-years after stroke (OR 0.66, 95% CI 0.46–0.92; P =  0.006).

Conclusions

BDNF gene variants were not major contributors to ischemic stroke severity, recovery, or short-term functional outcome. However, this study suggests that variants in the BDNF gene may contribute to poor long-term functional outcome after ischemic stroke.     

Population Genetic Variation in the Tree Fern Alsophila spinulosa (Cyatheaceae): Effects of Reproductive Strategy

Background

Essentially all ferns can perform both sexual and asexual reproduction. Their populations represent suitable study objects to test the population genetic effects of different reproductive systems. Using the diploid homosporous fern Alsophila spinulosa as an example species, the main purpose of this study was to assess the relative impact of sexual and asexual reproduction on the level and structure of population genetic variation.

Methodology/Principal Findings

Inter-simple sequence repeats analysis was conducted on 140 individuals collected from seven populations (HSG, LCH, BPC, MPG, GX, LD, and ZHG) in China. Seventy-four polymorphic bands discriminated a total of 127 multilocus genotypes. Character compatibility analysis revealed that 50.0 to 70.0% of the genotypes had to be deleted in order to obtain a tree-like structure in the data set from populations HSG, LCH, MPG, BPC, GX, and LD; and there was a gradual decrease of conflict in the data set when genotypes with the highest incompatibility counts were successively deleted. In contrast, in population ZHG, only 33.3% of genotypes had to be removed to achieve complete compatibility in the data set, which showed a sharp decline in incompatibility upon the deletion of those genotypes. All populations examined possessed similar levels of genetic variation. Population ZHG was not found to be more differentiated than the other populations.

Conclusions/Significance

Sexual recombination is the predominant source of genetic variation in most of the examined populations of A. spinulosa. However, somatic mutation contributes most to the genetic variation in population ZHG. This change of the primary mode of reproduction does not cause a significant difference in the population genetic composition. Character compatibility analysis represents an effective approach to separate the role of sexual and asexual components in shaping the genetic pattern of fern populations.     

遗传变异的又一来源：拷贝数变异

人们很早就发现DNA拷贝数变异与特定染色体重组和基因组异常相关这一现象,但最近才知道它与疾病的相关联系。我们对拷贝数变异的原理、最新研究方法,及其与复杂疾病的相关性研究等进展进行了综述;总结了拷贝数变异研究所存在的问题;对拷贝数变异未来的研究重点和需要解决的问题进行了展望。     

Adaptation,Clonal Interference,and Frequency-Dependent Interactions in a Long-Term Evolution Experiment with Escherichia coli

Twelve replicate populations of Escherichia coli have been evolving in the laboratory for >25 years and 60,000 generations. We analyzed bacteria from whole-population samples frozen every 500 generations through 20,000 generations for one well-studied population, called Ara−1. By tracking 42 known mutations in these samples, we reconstructed the history of this population’s genotypic evolution over this period. The evolutionary dynamics of Ara−1 show strong evidence of selective sweeps as well as clonal interference between competing lineages bearing different beneficial mutations. In some cases, sets of several mutations approached fixation simultaneously, often conveying no information about their order of origination; we present several possible explanations for the existence of these mutational cohorts. Against a backdrop of rapid selective sweeps both earlier and later, two genetically diverged clades coexisted for >6000 generations before one went extinct. In that time, many additional mutations arose in the clade that eventually prevailed. We show that the clades evolved a frequency-dependent interaction, which prevented the immediate competitive exclusion of either clade, but which collapsed as beneficial mutations accumulated in the clade that prevailed. Clonal interference and frequency dependence can occur even in the simplest microbial populations. Furthermore, frequency dependence may generate dynamics that extend the period of coexistence that would otherwise be sustained by clonal interference alone.     

中国部分地方黄牛品种线粒体D-loop区的遗传变异与进化分析

测定了13个黄牛品种125个个体的线粒体D-loop区段的全序列,包括12个中国地方黄牛品种的123个个体和德国黄牛2个个体,并进行了分析。结果显示,共检测到93个变异位点,57个单倍型,平均核苷酸差异(average number ofnucleotide differences,k)为22.708,核苷酸多样度(nucleotide diversity,π)为0.0251±0.00479,单倍型多样度(haplotypediversity,Hd)为0.888±0.026,表明我国黄牛品种遗传多样性非常丰富。构建的Neighbor-Joining进化树显示这13个品种主要分成两大类型:普通牛和瘤牛;新发现的特殊类型Ⅲ只有一个西藏阿沛甲咂牛的个体,它与牦牛D-loop序列最相近,证明西藏地区的黄牛与牦牛之间存在基因渗入现象。普通牛和瘤牛在日喀则驼峰牛中占的比例分别是64.3%和35.7%,在阿沛甲咂牛中占的比例分别是50.0%和50.0%,证明了西藏的黄牛也有瘤牛类型。云南牛品种的单倍型非常丰富证明了云南在中国黄牛起源上的重要地位;在27个中国黄牛品种中(本研究11个品种以及GenBank上的16个品种)找到了中国瘤牛的核心单倍型i1,并且对它进行了讨论。同时证明了西藏瘤牛独立于中国瘤牛核心类群的特殊性。     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996     

Variation in Reproductive Success Across Captive Populations: Methodological Differences,Potential Biases and Opportunities

Our understanding of fundamental organismal biology has been disproportionately influenced by studies of a relatively small number of ‘model’ species extensively studied in captivity. Laboratory populations of model species are commonly subject to a number of forms of past and current selection that may affect experimental outcomes. Here, we examine these processes and their outcomes in one of the most widely used vertebrate species in the laboratory – the zebra finch (Taeniopygia guttata). This important model species is used for research across a broad range of fields, partly due to the ease with which it can be bred in captivity. However despite this perceived amenability, we demonstrate extensive variation in the success with which different laboratories and studies bred their subjects, and overall only 64% of all females that were given the opportunity, bred successfully in the laboratory. We identify and review several environmental, husbandry, life‐history and behavioural factors that potentially contribute to this variation. The variation in reproductive success across individuals could lead to biases in experimental outcomes and drive some of the heterogeneity in research outcomes across studies. The zebra finch remains an excellent captive animal system and our aim is to sharpen the insight that future studies of this species can provide, both to our understanding of this species and also with respect to the reproduction of captive animals more widely. We hope to improve systematic reporting methods and that further investigation of the issues we raise will lead both to advances in our fundamental understanding of avian reproduction as well as to improvements in future welfare and experimental efficiency.