Morphometric analysis of dinosaur eggshells: constraints of size on shape

Studies on fossil eggs and eggshells have been traditionally focused on analysis of microstructure and systematic classification, but few attempts have been made to examine the variability of eggshell characters. This work examines the variability of one diagnostic character, shape of the calcitic units, of some dinosaur eggshells by using outline shape analysis (elliptic Fourier analysis). The studied material belongs to five different oospecies of the parafamily Megaloolithidae Zhao, 1979. To test shape variation within and between oospecies, an analysis on 129 outlines was carried out and their variability was summarised by principal component analysis. We then tested if the shape of megaloolithid units changes according to size (i.e. allometry). A regression analysis between eggshell unit shape and two different size measurements (centroid size and eggshell thickness) showed that the thinnest eggshells (thickness ≤ 1 mm) have different shapes than thicker ones ( ≥ 2.5 mm). Our results stress that the variability in the shape of eggshell units in Megaloolithus oospecies is correlated to size (i.e. eggshell thickness), a phenomenon that should be then considered carefully when determining oospecies, since a single oospecies can show both a wide range of eggshell thickness and combined with different eggshell unit shapes.     

Implications de l’analyse microstructurale et géochimique des œufs de dinosaures de la cairanne (Bassin d’Aix en Provence,France, Rognacien inférieur)

Microstructural and geochemical analyses of the dinosaur eggshells at La Cairanne (Aix en Provence Basin, Lower Rognacien) show the presence of diagenesis. Diagenetic processes have altered the thickness of the shells and some structural data. The chemical composition has been also modified. Thus it is difficult to use these parameters to reconstruct the paleoenvironment. It is possible, indeed probable, that the problems of the previous authors about the definitions of eggshell morphotypes in the Aix Basin are due to diagenetic variations.     

Sediment and eggshell interactions: using abrasion to assess transport in fossil eggshell accumulations

Recent excavation at the Late Cretaceous Egg Mountain locality in Montana revealed abundant Spheroolithus eggshell fragments. The depositional history of the fragments is problematic due to the vertical dispersal of eggshell in homogeneous mudstone. The results of previous studies assessing eggshell transport are based on thin, modern eggshell occurring on well-defined horizons. Therefore, the assessment of depositional history required a different method. Chicken (Gallus gallus domesticus) and ostrich (Struthio camelus) eggshells were placed in a tumbler with water and quartz sand for 168–504 h to simulate transport. The resulting wear on these fragments, revealed under scanning electron microscope, was compared to unabraded eggshell. In addition, the modern eggshell was compared to fossil eggshells from a nesting site, crevasse splay and channel deposits, and Egg Mountain. Fossil eggshells from high-energy paleoenvironments display similar edge rounding to modern eggshell placed in the tumbler. In contrast, eggshell from Egg Mountain lacked edge rounding and resembled fossil eggshell from a nesting site and unabraded modern eggshell, suggesting that the Egg Mountain locality represents a parautochthonous assemblage. This study indicates that sediment interaction leaves distinct patterns of abrasion on eggshell, which may be useful for assessing transport of eggshell at fossil localities.     

Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates

The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species.     

Great spotted cuckoo eggshell microstructure characteristics can make eggs stronger

Obligate avian brood parasites lay stronger eggs than their hosts or non‐parasitic relatives because they are rounder and have a thicker eggshell. Additionally, some other characteristics of the brood parasitic eggshells related to their microstructure such as size and orientation of calcite crystal units could also contribute to generating even stronger shells. An eggshell microstructure formed by small randomly oriented calcite crystal units can increase the robustness of the eggshells of birds. Here, the eggshell microstructure of avian brood parasites as well as their hosts have been characterized in detail, using X‐ray diffraction analyses to estimate the size and degree of orientation of calcite crystal units making the eggshell. Specifically, the brood parasitic great spotted cuckoo Clamator glandarius and two hosts (jackdaws Corvus monedula and magpie Pica pica) and one non‐host species (the pigeon, Columba livia domestica) were considered. Calcite crystal of the eggshell of the brood parasitic species was smaller and more randomly oriented than those of the eggshells of non‐parasitic species, which suggest that eggshell microstructure would contribute to explain why parasitic eggs are more resistant to breakage than those of their hosts.     

Two new theropod egg sites from the Late Jurassic Lourinhã Formation,Portugal

Two new Late Jurassic (uppermost Late Kimmeridgian) dinosaur eggshell sites are described, Casal da Rola and Porto das Barcas, both near Lourinhã, central-west Portugal. Casal da Rola yields eggshells with an obliquiprismatic morphotype comparable to those from a nest with the associated fossil embryos from Paimogo, tentatively assigned to the theropod Lourinhanosaurus antunesi. The Porto das Barcas eggshells have a dendrospherulitic morphotype with a prolatocanaliculate pore system. This morphotype was also recognised in eggshells from a clutch with associated Torvosaurus embryos at the Porto das Barcas locality. A preliminary cladistic analysis of eggshell morphology suggests theropod affinities for the Casal da Rola eggs, but is unable to resolve the phylogenetic position of the Porto das Barcas eggs. The eggshells at both sites are preserved in distal flood plain mudstones and siltstones. Carbonate concretions within the deposits indicate paleosol development.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Mapping of quantitative trait loci affecting eggshell quality on chromosome 9 in an F2 intercross between two chicken lines divergently selected for eggshell strength

Broken and cracked eggshells are major causes of significant economic losses to the egg production industry. The quantitative trait loci (QTL) on chromosome 9 influencing the quality of eggshells were identified by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation conducted over 14 generations. Chromosome-wide highly significant ( P  <   0.01) QTL associated with egg weight (EW), short length of egg (SLE), long length of egg (LLE) and eggshell weight were mapped to the distal region of chromosome 9. Among the QTL affecting EW, SLE and LLE, ovocalyxin-32 was identified as a potential candidate gene influencing eggshell traits. Marker-assisted selection based on these QTL could be used to develop strategies for reducing the breakage and cracking of eggs in commercial layer houses.     

Fossile Reptil-Eischalen (Chelonia,Crocodilia, Dinosauria) aus dem unteren Barremium von Galve (Provinz Teruel,SE-Spanien)

The Lower Cretaceous vertebrate bearing sediments of Galve (province of Teruel, SE Spain) have yielded some hundred fossil eggshell fragments, all slightly diagenetically altered. Concerning their microstructure, an assignment of the material to different taxa of reptiles was possible. The eggshell assemblage of Galve consists of a-c) three types of chelonian eggshells, one of them probably representing the subfamily Batagurinae, which would extend this taxon back into the Lower Cretaceous, d) the oldest known crocodilian eggshells, with lacunae systems on the outer surface, comparable to Recent taxa, and e-f) two different types of dinosaurian eggshells. In correspondence to the suggested fluviatile paleoenvironment of Galve, the fossil eggshells described herein are fragmented and represent mostly turtles and crocodiles.     

Structure of shells from eggs of kinosternid turtles

Shells from eggs of five species of kinosternid turtle (Sternotherus minor, Kinosternon flavescens, K. baurii, K. Hirtipes, and K. alamosae) were examined with light and scanning electron microscopy. Except for possible differences among species in thickness of eggshells, structure of shells from all eggs was similiar. In general, kinosternid turtles lay eggs having a rigid calcareous layer composed of calcium carbonate in the form of aragonite. The calcareous layer is organized into individual shell units with needlelike crystallites radiating from a common center. Most of the thickness of the eggshell is attributable to the calcareous layer, with the fibrous shell membrane comprising only a small fraction of shell thickness. Pores are found in the calcareous layer, but they are not numereous. The outer surface of the eggshells is sculptured and may have a thick, organic layer in places. The outer surface of the shell membrane of decalcified eggshells is studded with spherical cores which presumably nucleate growth of shell units during shell formation. The shell membrane detaches from eggs incubated to hatching, carrying with it remnants of the calcareous layer. Such changes in shell structure presumably reflect withdrawal of calcium from the eggshell by developing embryos.     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Structure-function relationship of avian eggshell matrix proteins: a comparative study of two major eggshell matrix proteins, ansocalcin and OC-17

The role of individual matrix proteins in avian eggshell calcification is poorly understood despite numerous attempts to characterize and localize their presence in the eggshell matrix. Ansocalcin, the major matrix protein from goose eggshell, was found to induce the formation of calcite crystal aggregates under in vitro. Owing to its high similarity with the chicken eggshell matrix protein ovocleidin 17 (OC-17), a comparative investigation has been carried out to understand the structure-function relationship. RP-HPLC shows that ansocalcin is the major component in extracts of goose eggshells before and after bleach treatment. However, OC-17 was observed in minute quantities in the extract of bleach-treated chicken eggshells. In vitro crystal growth experiments showed that OC-17 and ansocalcin interact differently with the calcite crystals formed. Circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering studies showed that, under the conditions used in our experiments, OC-17 does not aggregate in solution or induce the nucleation of calcite aggregates in the concentration range used. These observations indicate that OC-17 and ansocalcin play different roles in the eggshell calcification. To our knowledge, this is the first report on the comparison of properties of homologous eggshell proteins that belong to the same phylogeny.     

PROPERTIES OF AVIAN EGG SHELLS AND THEIR ADAPTIVE VALUE

I. An arbitrary classification of avian eggshells is proposed.
2. The role of the eggshell in conserving the water in eggs at oviposition is discussed. There is as yet no correlation between this property and the pore systems in avian eggshells.
3. The pore systems may act as diffusion pathways and the hypothesis has been advanced that in many eggs the shells are adapted so that restriction of gaseous diffusion by mud, preening oils and nest debris is prevented.
4. The mechanical properties of the shell are considered in the novel context of defence against (a) attrition that could lead to the pores being blocked with dust, and (b) cracking that would destroy the diffusion pathways noted in 3.
5. The overall objective of the review was to discuss the concept that avian eggshell are adapted to fit an egg to the nest environment.     

Global diversity and adaptations of avian eggshell thickness indices

The amniote eggshell is a fundamental aspect of the embryo life-support system, protecting it from UV light, microbes and mechanical damage, while regulating gas exchange and providing calcium for growth. The thickness of eggshells is highly diverse across modern birds and influences multiple eggshell functions, yet the selective pressures driving eggshell thickness have not been clearly identified. Here, we use a global dataset of avian eggshell thickness indices for 4260 (> 41%) avian species to assess trends in eggshell thickness across the phylogeny, as these indices are strongly correlated with direct measures of eggshell thickness and are non-destructive to the sample. We analysed the dataset within a phylogenetic framework to assess the relative importance of climatic and ecological explanations for variation in eggshell thickness indices. The distribution of avian eggshell thickness indices across species was found to be primarily driven by phylogenetic relatedness, in addition to evolutionary processes that do not match a Brownian model of evolution. Across modern birds, thicker eggshells were more prevalent in species (1) with precocial young, (2) which exhibit a scavenger-based diet, (3) which primarily feed on vertebrates or plants (excluding nectivores, seed and fruit specialists) and (4) which breed in open habitats. Thicker eggshells found in species with precocial young probably enable higher rates of calcium removal for the more advanced embryo development. Excessive light transmission through the shell damages developing embryos, while too little light transmission can impede development. Eggs in shaded habitats experience low light exposure, and thus thinner shells are more prevalent in these environments potentially to increase light transmission through the shell. Overall, variation in eggshell thickness indices appears to be driven largely by phylogeny, with certain life-history traits linked to embryo growth rate, calcium content of their food, and the need to mitigate excessive light transmission through the shell.     

Egg colouration predicts brood size,telomere length and body condition of spotless starling fledglings

Understanding the impressive interspecific variation in avian eggshell colouration has attracted the attention of evolutionary ecologists for more than a century. Several functional explanations predict positive covariation between eggshell pigmentation and phenotypic quality of nestlings. We test this prediction in spotless starlings Sturnus unicolor by using biometric measurements and telomere length of hatchlings and fledglings as proxies of phenotypic quality. Female spotless starlings lay immaculate blue‐green eggs, a sexually selected signal directed to males. Pigmentation predicts positive associations with concentration of antioxidants and testosterone in the yolk and with paternal provisioning effort during nestling growth. Eggshell pigmentation (blue‐green chroma) is not associated with telomere length of hatchlings, which suggests weak maternal effects on this trait. However, we find negative associations of eggshell colouration with both body condition and telomere length of fledglings. Moreover, we find positive associations between eggshell colouration and clutch size, which suggests that sibling competition is higher in nests with more coloured eggshells. Previous works demonstrated that level of sibling competition is positively related to telomere erosion and, thus, the detected negative associations between eggshell colouration, body condition and telomere length of fledglings would reflect higher level of competition in nests with more coloured eggshells. We therefore speculate with the possibility that females that lay larger clutches also lay more coloured eggshells that elicit increased paternal provisioning effort and, thus, raise larger broods at the expense of telomere erosion of their offspring.     

Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research.     

The evolution of size of the uropygial gland: mutualistic feather mites and uropygial secretion reduce bacterial loads of eggshells and hatching failures of European birds

Potentially, pathogenic bacteria are one of the main infective agents against which a battery of chemical and physical barriers has evolved in animals. Among these are the secretions by the exocrine uropygial gland in birds. The antimicrobial properties of uropygial secretions may prevent colonization and growth of microorganisms on feathers, skin and eggshells. However, uropygial gland secretions also favour the proliferation of feather mites that feed on secretions and microorganisms living on feathers that would otherwise reach eggshells during incubation if not consumed by feather mites. Therefore, at the interspecific level, uropygial gland size (as an index of volume of uropygial secretion) should be positively related to eggshell bacterial load (i.e. the risk of egg infection), whereas eggshell bacterial loads may be negatively related to abundance of feather mites eating bacteria. Here, we explore these previously untested predictions in a comparative framework using information on eggshell bacterial loads, uropygial gland size, diversity and abundance of feather mites and hatching success of 22 species of birds. The size of the uropygial gland was positively related to eggshell bacterial loads (mesophilic bacteria and Enterobacteriaceae), and bird species with higher diversity and abundance of feather mites harboured lower bacterial density on their eggshells (Enterococcus and Staphylococcus), in accordance with the hypothesis. Importantly, eggshell bacterial loads of mesophilic bacteria, Enterococcus and Enterobacteriaceae were negatively associated with hatching success, allowing us to interpret these interspecific relationships in a functional scenario, where both uropygial glands and mutualistic feather mites independently reduce the negative effects of pathogenic bacteria on avian fitness.     

Palaeoenvironmental reconstruction of dinosaur nesting sites based on a geochemical approach to eggshells and associated palaeosols (Maastrichtian, Provence Basin, France)

Variability of the stable isotopic (δ¹³C and δ¹⁸O) and trace element (Mg, Sr, Fe, and Mn) content from dinosaur eggshells from the Provence Basin has been addressed by considering the stratigraphic distribution of the eggshells, the palaeoenvironments, and the chemical content of associated sediments. Preferentially, in situ eggshells were analysed. Samples were collected from two sections, one belonging to the floodplain (Roques Hautes) and the other to the channel belt (Rousset-Bréguières), as determined from sedimentological criteria. On a regional scale, this combined analysis provided new insights into palaeohydrology, vegetation cover and dinosaur behaviour. Different sources of drinking water were identified: (1) the main stream waters with significant salinity levels, (2) lateral feeders with water composition close to that of precipitation. Water composition is reflected in the eggshell trace element contents: a high Sr content for the eggshells from the channel belt (mean 1200 ppm) contrasting with the very low values for the floodplain (mean 250 ppm). During the first part of the Maastrichtian, evaporation rates were quite significant, as seen from the 1.5‰ difference between the eggshell δ¹⁸O (drinking water) and the palaeosol carbonate nodules (precipitation). This contrast diminished and became nearly insignificant in the second half of the Maastrichtian. Vegetation was composed of C₃ plants and two ecosystems are reconstructed: a riparian forest extending along the channel belt and open vegetation in the floodplain. The contrasted isotopic and trace element content of the eggshells was a crucial factor in identifying animal migration from one site to another. Migration was quite limited, suggesting that the animals settled for some time at the places where they laid their eggs. Intraspecific variations among modern birds are well correlated with the environment and may be greater than interspecies variations, which limits the potential of geochemical analysis to characterise dinosaur species.     

三种海鸟卵壳的超微结构和无机成分的研究

应用扫描电镜和ＴＮ－５５００能谱仪对三种海鸟卵壳的超微结构和基本无机元素进行研究分析,结果表明,基本结构相似,但表层突起和裂纹,乳锥和锥本形态,以及气孔在单位面积内的数量,壳膜与卵壳的锚连方式,壳膜元素组成等都存在着不同,这既反映其遗传,繁殖生理功能上的差异,也显示了不同种鸟卵壳之间的相似和相异性,因而对研究严分类和地理分布具有一定差异价值。     

Electron backscatter diffraction (EBSD) analysis of maniraptoran eggshells with important implications for microstructural and taphonomic interpretations

Electron backscatter diffraction (EBSD) is a useful tool for gathering crystallographic information from carbonate biominerals because it maps out the orientation of crystal grains very precisely. EBSD has become popular in invertebrate palaeontology but its application in vertebrate palaeontology remains limited. However, the study of fossil eggshells is a field where EBSD has wide potential applicability and provides a quantitative approach to fossil eggshell research as well as new qualitative data. Here we analyse fossil and extant maniraptoran dinosaur eggshells using EBSD analysis emphasizing four different aspects. The mapping imaging clarifies previously ambiguous characters such as squamatic ultrastructure and allows a more objective evaluation of avian and non‐avian maniraptoran eggshell. In particular, our results imply that the presence of an external zone in the manirpatoran eggshell is not diagnostic of avian eggshell. EBSD analysis can be also used for differentiating true pore canals from cracks in the eggshell radial section, thereby determining the biological genuineness or otherwise of a pore‐like structure. Finally, the misorientation angle distribution of the material shows a clear dichotomy that may reflect reproductive brooding strategy, although further studies on contact incubation of palaeognaths and neognaths are needed to confirm this.