Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests with fully exposed eggs only became widespread among Euornithes. A potential co-evolution of open nests and brooding behavior among maniraptorans may have freed theropods from the ground-based restrictions inherent to covered nests and allowed the exploitation of alternate nesting locations. These changes in nesting styles and behaviors thus may have played a role in the evolutionary success of maniraptorans (including birds).

Copyright: © 2015 Tanaka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

As a viable alternative to the water vapor conductance method, we present a statistically rigorous approach using eggshell porosity in order to predict nest type in extinct archosaurs. We apply this approach to the eggs of a variety of dinosaurs, including titanosaurs, the theropod Lourinhanosaurus, oviraptorosaurs, and troodontids, in order to assess their nesting habits and discuss the evolution of nest type and incubation behaviors among archosaurs.

Most prior studies have used a method that estimates the diffusive capacity of the eggshell, referred to as water vapor conductance (i.e.,G H2O ), to infer the types of nest built by dinosaurs. Water vapor conductance in living archosaurs has usually been measured experimentally via daily water loss of a fresh egg (e.g., [ 12 , 13 ]). A theoretical formula to calculate water vapor conductance from eggshell porosity was also developed based on Fick's law of diffusion (herein referred to as morphometric G H2O ) [ 12 ]. This formula was used initially by Seymour [ 11 ] to calculate G H2O for dinosaur eggs, and nest type was inferred on the premise that covered nests are found in living species with high G H2O and open nests in species with low G H2O values. Thus, morphometric G H2O values of dinosaurs were compared directly with experimental G H2O values of living archosaurs (e.g., [ 11 , 14 – 17 ]), although the latter were calculated from measurement of daily water loss of an egg (i.e., experimental G H2O ) and not from the theoretical formula (i.e., morphometric G H2O ). However, a recent study compared morphometric and experimental G H2O values in living archosaur species, and demonstrated that these two datasets/methods are mutually incongruent, likely due to systematic errors [ 18 ]. Thus direct comparison between morphometric and experimental G H2O values, as widely applied to infer nest type of dinosaurs, may not be valid.

Nest types and associated nesting behaviors are poorly understood in extinct archosaurs, including non-avian dinosaurs, in part because nest structures and nesting materials are rarely preserved [ 6 , 7 ]. Even on the rare occasions where nest structures are found (e.g., excavations, mounds; [ 7 – 10 ]), there is no indication of whether the eggs were covered by organic/inorganic material or surrounded by nesting materials as typically found in living archosaurs. Consequently, other evidence related to egg clutches, such as their taphonomic and sedimentologic setting or eggshell structures (i.e., pore canals), have been used to infer the nest type of dinosaurs (e.g., [ 7 , 11 ]).

Nests are varied structures that play an important role in archosaur biology because they are used for incubating eggs and, in many species, for raising young. The nests can consist of simple scrapes or holes in the ground, bowl-shaped structures, or large vegetation mounds [ 1 , 2 ], and their architecture is suited not only for the incubation of eggs in a given environment but also for the incubation behavior/method of a species. Among extant archosaurs, two general types of nest are observed: 1) covered nests, in which the eggs are covered by organic/inorganic matter, are built by species that incubate their eggs using external heat sources (e.g., solar heat, plant decomposition, or geothermal heat [ 3 ]), and 2) open nests, in which the eggs are not covered by substrate and left exposed, are built by species that brood their eggs. Because all crocodylian species build covered nests and all bird species, except those of megapodes, incubate eggs in open nests [ 4 ], the transition from covered to open nest type likely occurred among non-avian dinosaurs (e.g., [ 5 ]).

Material and Methods

A series of methodological steps were taken to document the relationship between eggshell porosity and nest types in crocodylians and birds in order to infer nest types in extinct archosaurs. Data on eggshell porosity (A p ∙L s -1, in mm), egg mass (M, in g), and nest types (see "Nest classification for extant taxa") were compiled for living crocodylians and birds and subsequently compared statistically to test whether eggshell porosity relative to egg mass differs between open and covered nest types. Eggshell porosity and egg mass were then estimated for a variety of extinct archosaurs, including crocodylomorphs, non-avian dinosaurs, and birds. Through comparison with the extant dataset, discriminant analyses were used to infer nest types in extinct taxa. Both phylogenetic and non-phylogenetic (i.e., conventional) approaches were applied for the statistical analyses.

Relationship between Water Vapor Conductance and Eggshell Porosity Water vapor conductance of living species has usually been measured experimentally (e.g., [12,13]), but it has also been shown to be related to the geometry of eggshell pore canals. Ar et al. [12] were the first to derive a mathematical equation to calculate morphometric water vapor conductance (G H2O ) using pore geometry in archosaur eggs. This equation is expressed as: (1) where c is a unit conversion constant (1.56 x 109 mgH 2 O∙s∙day-1∙mol-1), D H2O is the diffusion coefficient of water vapor (mm2∙s-1) in air, R is the universal gas constant (6.24 x 107 mm3∙torr∙mol-1°K-1), T is the absolute temperature of incubation (°K), A p ∙L s -1 is eggshell porosity, A p is the total pore area of an egg (mm2), and L s is pore length (mm) [12]. Since many variables (i.e., c, D H2O , R, and T) can be safely assumed to be consistent among species (e.g., [12,13,18]), the equation can be simplified and expressed as G H2O = 2.1∙A p ∙L s -1 (see [18]). Morphometric water vapor conductance is thus directly proportional to eggshell porosity. Given that morphometric water vapor conductance (and hence eggshell porosity) is influenced by absolute nest humidity (S1 Text), which in turn is correlated with nest architecture or type (covered vs. open, see [19]), a correlation between eggshell porosity and nest types can be sought (see S1 Text for further explanation).

Selection of extant taxa Eggshell porosity, egg mass and nest type for 127 species of extant birds and crocodylians were gathered from either the literature (see [18]) or via new measurements of egg specimens (S2, S3 and S4 Tables). Egg specimens were permitted to be accessed from the institutions listed in S2 Table. The dataset includes only species with pore canals that approximate simple (unbranched) or tubular structures because porosity of eggshells with more complex pores (e.g., branched pores) could not be accurately estimated (e.g., Casuarius, Dromaius, Pterocnemia, Rhea, and Struthio; [20–23]). Although some pores of crocodylian eggshells may be irregularly shaped, they are usually simple and straight [24] and are here assumed to be tubular.

Nest classification for extant taxa Nest structures of extant archosaurs were classified into two general types, covered nests and open nests, based on information available in the literature (S4 Table). Covered nests are defined as those in which the eggs are completely covered with vegetation and/or sediment (e.g., mound or infilled hole nests on/in the ground), whereas open nests are those in which the eggs are partly or fully exposed, and may have nest materials surrounding a portion of the eggs (e.g., scrape, cup, plate, and dome nests) (see [19]). Certain aquatic birds (e.g., Podicipediformes, Cygnus, Oxyura, Chlidonias niger, and Gavia immer) were excluded from this study due to their unusual nesting style. Because these birds build open nests floating on water with nest materials that can be wet [25–29], presumably resulting in high nest humidity [19,25–27], their eggshell porosity and water vapor conductance are anomalously high for birds with open nests [13,26,30].

Selection of fossil eggs/ootaxa Eggshell porosity and egg mass for 29 extinct archosaur taxa and ootaxa (i.e., egg taxa) were compiled from the literature or from new/additional measurements of egg specimens listed in S2 Table. Only species and oospecies with simple pore canals and for which data for individual pore area, pore density, pore length, and egg length and breadth were available were included in this study (Table 1). Eggs and ootaxa with complex or irregular pores {e.g., Dendroolithidae (Torvosaurus and possibly therizinosaur), Faveoloolithidae (?Sauropodomorpha), Ovaloolithidae (?Ornithopoda), and Spheroolithidae (Maiasaura-like eggs, presumably hadrosaur)} and taxa/ootaxa for which the data were potentially derived from the combination of multiple oospecies (e.g. 'Hypselosaurus' and 'Protoceratops' in [11] and Elongatoolithidae in [31,32]) were not included in this study. Also, eggshell porosity for enantiornithine eggs (e.g. 'Gobipteryx minuta' in [31]) was not estimated in our study because the original article [31] indicated questionable values for both total number of pores and individual pore area. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. List of extinct archosaur taxa/ootaxa with estimated egg mass (M) and eggshell porosity (A p ∙Ls-1) used in this study. https://doi.org/10.1371/journal.pone.0142829.t001 The taxonomic affinity of most ootaxa considered in this study is well-established, particularly at higher taxonomic levels. For example, the ootaxon Bauruoolithus is attributed to a crocodylomorph based on eggshell microstructure [33]. The ootaxon Megaloolithus patagonicus is referred to a titanosaur based on its association with embryonic remains [49,50], thus eggs of the Megaloolithidae oofamily are widely regarded as belonging to sauropods [49,51,52]. Formerly classified in Megaloolithidae, the ootaxon Cairanoolithus has recently been re-assigned to a new oofamily, Cairanoolithidae, by Sellés and Galobart [53] who suggested it may belong to an ornithischian dinosaur. The taxonomic identity of some theropod eggs is also known based on association with either embryonic or parental skeletal remains. These include the eggs of Lourinhanosaurus antunesi, a large theropod of either allosauroid [54,55] or coelurosaurian [56] affinity, the ootaxon Macroolithus yaotunensis, assigned to an oviraptorosaur [57], and the ootaxon Prismatoolithus levis, assigned to Troodon formosus [58]. Elongatoolithid and prismatoolithid ootaxa are attributed to Oviraptorosauria [57,59–64] and non-oviraptorosaur maniraptorans [46,65], respectively, based on eggshell microstructure similarities with eggs of known taxonomic identity. The ootaxon Continuoolithus is assigned to an indeterminate theropod based on egg and eggshell morphology [9,39,66]. Moa eggshells have been assigned to two small-bodied (female body masses 20–30 kg) species, Pachyornis geranoides and Euryapteryx sp., based on DNA analyses [48].

Egg mass Mean egg mass (M, in g) for living and extinct archosaurs was compiled for this study. Egg mass for living species was obtained from the literature (S3 Table) and that for fossil taxa/ootaxa was estimated from egg length and breadth using the equation of Hoyt [68] (Table 3). Although other methods exist to estimate fossil egg mass (see [17]), they produce results that are consistent (within 10%) with Hoyt’s [68] method [17,39]. Therefore, for consistency, we applied Hoyt's method to all extinct ootaxa/taxa. Egg mass for Elongatoolithus andrewsi, Macroolithus rugustus, and M. yaotunensis was taken from Mou [41], who had used Hoyt's [68] method to derive his estimates.

Phylogenetic distribution of nest type The nature of the phylogenetic distribution (i.e., random vs. clumped) of nest types among living archosaurs was investigated based on our compiled extant dataset. Because nest type can be coded as a binary trait (covered vs. open), Fritz and Purvis' [72] D statistic was calculated by running 1000 permutations of the 'phylo.d' function of the package 'caper' using the software platform R3.1.3 (http://www.r-project.org/). For the D statistic, a value equal to or higher than 1.0 indicates a random phylogenetic distribution, whereas a value equal to or lower than 0 indicates a non-random phylogenetic distribution (i.e., phylogenetically clumped). The 'phylo.d' function provides p values to indicate whether the estimated D statistic is significantly different from 0 and 1, respectively. The D statistic was run using a phylogenetic tree of 127 species of living birds and crocodylians compiled from the large-scale phylogeny of Jarvis et al. [73] and other publications for small-scale interrelationships (S3 Fig). Branch length was estimated from the divergence times of each node following the procedures of Motani and Schmitz [74] and Schmitz and Motani [75]. Divergence times of major clades were obtained from Time Tree (http://timetree.org) for birds and from Oaks [76] for crocodylians. Terminal taxon ages were set to zero. The phylogenetic tree and character matrix were constructed with the PDAP module v.1.16 [77] of the software Mesquite 3.02 [78].

Analysis of covariance Eggshell porosity relative to egg mass was compared between extant open-nesting and covered-nesting archosaurs using both conventional and phylogenetically-corrected analysis of covariance (ANCOVA and pcANCOVA, respectively). Non-phylogenetic, ordinary least-squares regression (OLS) was implemented for conventional ANCOVA with IBM SPSS Statistics v. 22.0.0 (IBM SPSS Inc.), whereas phylogenetically-corrected ANCOVA was implemented with the MATLAB (MathWorks Inc.) program Regressionv2.m (available upon request from T. Garland Jr.) following the method of Lavin et al. [79]. A phylogenetic variance-covariance matrix for Regressionv2.m was generated with the DOS PDDIST program [80]. Regressions for pcANCOVA were generated with two evolutionary models: regressions with Brownian motion (PGLS) and Ornstein-Uhlenbeck models (RegOU). PGLS assumes an evolutionary process with "random walk in continuous time" (e.g., [79]), whereas RegOU assumes an evolutionary process of "wandering back and forth on a selective peak" [79,81,82]. These three regression models (OLS, PGLS, and RegOU) were compared using the Akaike Information Criterion (AIC) to determine the best fit model of regression, where a lower AIC value indicates a better fit (e.g., [79,83,84]). Nest type (open and covered nests) was considered a categorical variable, a covariate of egg mass, and a dependent variable of eggshell porosity in these analyses. Values of eggshell porosity (A p ∙L s -1) and egg mass (M) were log-10 transformed prior to analysis. The normality and homogeneity of variances of the dataset were tested by non-phylogenetic Shapiro-Wilk tests and Levene tests using IBM SPSS Statistics v. 22.0.0. Residuals of log A p ∙L s -1, calculated from OLS regressions for each nest type, were used for the Shapiro-Wilk tests. The phylogenetic tree compiled for the D statistic (see above) was used for the pcANCOVA. In addition to the branch length determination method based on divergence time used for the D statistic, an arbitrary standardized method was also applied to assign branch length for the pcANCOVA because branches were not adequately standardized by divergence time. An arbitrary branch length model was used by following the procedure of Garland et al. [85], resulting in all branch lengths equal to one.