Nonpathological inflammation drives the development of an avian flight adaptation.

The development of modern birds provides a window into the biology of their dinosaur ancestors. We investigated avian postnatal development and found that sterile inflammation drives formation of the pygostyle, a compound structure resulting from bone fusion in the tail. Inflammation is generally induced by compromised tissue integrity, but here is involved in normal bone development. Transcriptome profiling and immuno/histochemistry reveal a robust inflammatory response that resembles bone fracture healing. The data suggest the involvement of necroptosis and multiple immune cell types, notably heterophils (the avian equivalent of neutrophils). Additionally, nucleus pulposus structures, heretofore unknown in birds, are involved in disc remodeling. Anti-inflammatory corticosteroid treatment inhibited vertebral fusion, substantiating the crucial role of inflammation in the ankylosis process. This study shows that inflammation can drive developmental skeletogenesis, in this case leading to the formation of a flight-adapted tail structure on the evolutionary path to modern avians.

A remarkable group of thick-headed Triassic Period archosauromorphs with a wide, possibly Pangean distribution.

The radiation of archosauromorph reptiles in the Triassic Period produced an unprecedented collection of diverse and disparate forms with a mix of varied ecologies and body sizes. Some of these forms were completely unique to the Triassic, whereas others were converged on by later members of Archosauromorpha. One of the most striking examples of this is with Triopticus primus, the early dome-headed form later mimicked by pachycephalosaurid dinosaurs. Here we fully describe the cranial anatomy of Triopticus primus, but also recognize a second dome-headed form from a Upper Triassic deposit in present-day India. The new taxon, Kranosaura kuttyi gen. et sp. nov., is likely the sister taxon of Triopticus primus based on the presence of a greatly expanded skull roof with a deep dorsal opening (possibly the pineal opening) through the dome, similar cranial sculpturing, and a skull table that is expanded more posterior than the posterior extent of the basioccipital. However, the dome of Kranosaura kuttyi gen. et sp. nov. extends anterodorsally, unlike that of any other archosauromorph. Histological sections and computed tomographic reconstructions through the skull of Kranosaura kuttyi gen. et sp. nov. further reveal the uniqueness of the dome of these early archosauromorphs. Moreover, our integrated analysis further demonstrates that there are many ways to create a dome in Amniota. The presence of 'dome-headed' archosauromorphs at two localities on the western and eastern portions of Pangea suggests that these archosauromorphs were widespread and are likely part of more assemblages than currently recognized.

Evolutionary trends in Triceratops from the Hell Creek Formation, Montana.

The placement of over 50 skulls of the well-known horned dinosaur Triceratops within a stratigraphic framework for the Upper Cretaceous Hell Creek Formation (HCF) of Montana reveals the evolutionary transformation of this genus. Specimens referable to the two recognized morphospecies of Triceratops, T. horridus and T. prorsus, are stratigraphically separated within the HCF with the T. prorsus morphology recovered in the upper third of the formation and T. horridus found lower in the formation. Hypotheses that these morphospecies represent sexual or ontogenetic variation within a single species are thus untenable. Stratigraphic placement of specimens appears to reveal ancestor-descendant relationships. Transitional morphologies are found in the middle unit of the formation, a finding that is consistent with the evolution of Triceratops being characterized by anagenesis, the transformation of a lineage over time. Variation among specimens from this critical stratigraphic zone may indicate a branching event in the Triceratops lineage. Purely cladogenetic interpretations of the HCF dataset imply greater diversity within the formation. These findings underscore the critical role of stratigraphic data in deciphering evolutionary patterns in the Dinosauria.

Comparative histology of some craniofacial sutures and skull-base synchondroses in non-avian dinosaurs and their extant phylogenetic bracket.

Sutures and synchondroses, the fibrous and cartilaginous articulations found in the skulls of vertebrates, have been studied for many biological applications at the morphological scale. However, little is known about these articulations at the microscopic scale in non-mammalian vertebrates, including extant archosaurs (birds and crocodilians). The major goals of this paper were to: (i) document the microstructure of some sutures and synchondroses through ontogeny in archosaurs; (ii) compare these microstructures with previously published sutural histology (i.e. that of mammals); and (iii) document how these articulations with different morphological degrees of closure (open or obliterated) appear histologically. This was performed with histological analyses of skulls of emus, American alligators, a fossil crocodilian and ornithischian dinosaurs (hadrosaurids, pachycephalosaurids and ceratopsids). Emus and mammals possess a sutural periosteum until sutural fusion, but it disappears rapidly during ontogeny in American alligators. This study identified seven types of sutural mineralized tissues in extant and extinct archosaurs and grouped them into four categories: periosteal tissues; acellular tissues; fibrous tissues; and intratendinous tissues. Due to the presence of a periosteum in their sutures, emus and mammals possess periosteal tissues at their sutural borders. The mineralized sutural tissues of crocodilians and ornithischian dinosaurs are more variable and can also develop via a form of necrosis for acellular tissues and metaplasia for fibrous and intratendinous tissues. It was hypothesized that non-avian dinosaurs, like the American alligator, lacked a sutural periosteum and that their primary mode of ossification involved the direct mineralization of craniofacial sutures (instead of intramembranous ossification found in mammals and birds). However, we keep in mind that a bird-like sutural microstructure might have arisen within non-avian saurichians. While synchondroseal histology is relatively similar in archosaurs and mammals, the microstructural differences between the sutures of these two clades are undeniable. Moreover, the current results suggest that the degree of sutural closure can only accurately be known via microstructural analyses. This study sheds light on the microstructure and growth of archosaurian sutures and synchondroses, and reveals a unique, undocumented histological diversity in non-avian dinosaur skulls.

An Ion-exchange Bone Demineralization Method for Improved Time, Expense, and Tissue Preservation.

Here, we describe an ethylenediaminetetraacetic acid (EDTA)-based bone demineralization procedure that uses cation-exchange resin and dialysis tubing. This method does not require solution changes or special equipment, is faster than EDTA alone, is cost-effective, and is environmentally friendly. Like other EDTA-based methods, this procedure yields superior tissue preservation than formic acid demineralization. Greater protein antigenicity using EDTA as opposed to formic acid has been described, but we also find significant improvements in carbohydrate-based histological staining. Histological staining using this method reveals cartilage layers that are not distinguishable with formic acid demineralization. Carbohydrate preservation is relevant to many applications of bone demineralization, including the assessment of osteoarthritis from bone biopsies and the use of demineralized bone powder for tissue culture and surgical implants. The improvements in time, expense, and tissue quality indicate this method is a practical and often superior alternative to formic acid demineralization.

Evidence of proteins, chromosomes and chemical markers of DNA in exceptionally preserved dinosaur cartilage.

A histological ground-section from a duck-billed dinosaur nestling (Hypacrosaurus stebingeri) revealed microstructures morphologically consistent with nuclei and chromosomes in cells within calcified cartilage. We hypothesized that this exceptional cellular preservation extended to the molecular level and had molecular features in common with extant avian cartilage. Histochemical and immunological evidence supports in situ preservation of extracellular matrix components found in extant cartilage, including glycosaminoglycans and collagen type II. Furthermore, isolated Hypacrosaurus chondrocytes react positively with two DNA intercalating stains. Specific DNA staining is only observed inside the isolated cells, suggesting endogenous nuclear material survived fossilization. Our data support the hypothesis that calcified cartilage is preserved at the molecular level in this Mesozoic material, and suggest that remnants of once-living chondrocytes, including their DNA, may preserve for millions of years.

Growing up Tyrannosaurus rex: Osteohistology refutes the pygmy "Nanotyrannus" and supports ontogenetic niche partitioning in juvenile Tyrannosaurus.

Despite its iconic status as the king of dinosaurs, Tyrannosaurus rex biology is incompletely understood. Here, we examine femur and tibia bone microstructure from two half-grown T. rex specimens, permitting the assessments of age, growth rate, and maturity necessary for investigating the early life history of this giant theropod. Osteohistology reveals these were immature individuals 13 to 15 years of age, exhibiting growth rates similar to extant birds and mammals, and that annual growth was dependent on resource abundance. Together, our results support the synonomization of "Nanotyrannus" into Tyrannosaurus and fail to support the hypothesized presence of a sympatric tyrannosaurid species of markedly smaller adult body size. Our independent data contribute to mounting evidence for a rapid shift in body size associated with ontogenetic niche partitioning late in T. rex ontogeny and suggest that this species singularly exploited mid- to large-sized theropod niches at the end of the Cretaceous.

Distal spinal nerve development and divergence of avian groups.

The avian transition from long to short, distally fused tails during the Mesozoic ushered in the Pygostylian group, which includes modern birds. The avian tail embodies a bipartite anatomy, with the proximal separate caudal vertebrae region, and the distal pygostyle, formed by vertebral fusion. This study investigates developmental features of the two tail domains in different bird groups, and analyzes them in reference to evolutionary origins. We first defined the early developmental boundary between the two tail halves in the chicken, then followed major developmental structures from early embryo to post-hatching stages. Differences between regions were observed in sclerotome anterior/posterior polarity and peripheral nervous system development, and these were consistent in other neognathous birds. However, in the paleognathous emu, the neognathous pattern was not observed, such that spinal nerve development extends through the pygostyle region. Disparities between the neognaths and paleognaths studied were also reflected in the morphology of their pygostyles. The ancestral long-tailed spinal nerve configuration was hypothesized from brown anole and alligator, which unexpectedly more resembles the neognathous birds. This study shows that tail anatomy is not universal in avians, and suggests several possible scenarios regarding bird evolution, including an independent paleognathous long-tailed ancestor.

Ontogenetic changes in the long bone microstructure in the nine-banded armadillo (Dasypus novemcinctus).

Analysis of ontogenetic changes in long bone microstructure aid in vertebrate life history reconstructions. Specifically, osteohistological examination of common fauna can be used to infer growth strategies of biologically uncommon, threatened, or extinct vertebrates. Although nine-banded armadillo biology has been studied extensively, work on growth history is limited. Here we describe long bone microstructure in tibiae and femora of a limited ontogenetic series of nine- banded armadillos (Dasypus novemcinctus) to elucidate patterns of bone growth. The cortex of the smallest individual is composed of compacted coarse cancellous bone (CCCB) and woven tissue. Extensive cortical drift is driven by periosteal erosion and further compaction of trabeculae resulting in an increase in the amount of CCCB. The cortex of the largest specimens is primarily CCCB with thickened endosteal bone and thin outer cortices of lamellar and parallel-fibered tissue. The outer cortices of the largest individuals are interpreted as an external fundamental system (EFS) indicating a cessation of appositional bone growth corresponding to skeletal maturity (i.e. asymptotic or adult size). The EFS forms in femora prior to tibiae, indicating femoral growth rates begin decreasing earlier than tibial in D. novemcinctus. Growth trends in common fauna like the nine-banded armadillo can be used as a foundation for understanding life histories of related, but uncommon or extinct, species of cingulates.

Relative growth rates of predator and prey dinosaurs reflect effects of predation.

Hadrosaurs grew rapidly, and quantifying their growth is key to understanding life-history interactions between predators and prey during the Late Cretaceous. In this study, we longitudinally sampled a sequence of lines of arrested growth (LAGs) from an essentially full-grown hadrosaur Hypacrosaurus stebingeri (MOR 549). Spatial locations of LAGs in the femoral and tibial transverse sections of MOR 549 were measured and circumferences were calculated. For each bone, a time series of circumference data was fitted to several stochastic, discrete growth models. Our results suggest that the femur and the tibia of this specimen of Hypacrosaurus probably followed a Gompertz curve and that LAGs reportedly missing from early ontogeny were obscured by perimedullary resorption. In this specimen, death occurred at 13 years and took approximately 10-12 years to reach 95 per cent asymptotic size. The age at growth inflection, which is a proxy for reproductive maturity, occurred at approximately 2-3 years. Comparisons with several small and large predatory theropods reveal that MOR 549 grew faster and matured sooner than they did. These results suggest that Hypacrosaurus was able to partly avoid predators by outgrowing them.

Avian tail ontogeny, pygostyle formation, and interpretation of juvenile Mesozoic specimens.

The avian tail played a critical role in the evolutionary transition from long- to short-tailed birds, yet its ontogeny in extant birds has largely been ignored. This deficit has hampered efforts to effectively identify intermediate species during the Mesozoic transition to short tails. Here we show that fusion of distal vertebrae into the pygostyle structure does not occur in extant birds until near skeletal maturity, and mineralization of vertebral processes also occurs long after hatching. Evidence for post-hatching pygostyle formation is also demonstrated in two Cretaceous specimens, a juvenile enantiornithine and a subadult basal ornithuromorph. These findings call for reinterpretations of Zhongornis haoae, a Cretaceous bird hypothesized to be an intermediate in the long- to short-tailed bird transition, and of the recently discovered coelurosaur tail embedded in amber. Zhongornis, as a juvenile, may not yet have formed a pygostyle, and the amber-embedded tail specimen is reinterpreted as possibly avian. Analyses of relative pygostyle lengths in extant and Cretaceous birds suggests the number of vertebrae incorporated into the pygostyle has varied considerably, further complicating the interpretation of potential transitional species. In addition, this analysis of avian tail development reveals the generation and loss of intervertebral discs in the pygostyle, vertebral bodies derived from different kinds of cartilage, and alternative modes of caudal vertebral process morphogenesis in birds. These findings demonstrate that avian tail ontogeny is a crucial parameter specifically for the interpretation of Mesozoic specimens, and generally for insights into vertebrae formation.

Soft tissue and cellular preservation in vertebrate skeletal elements from the Cretaceous to the present.

Soft tissues and cell-like microstructures derived from skeletal elements of a well-preserved Tyrannosaurus rex (MOR 1125) were represented by four components in fragments of demineralized cortical and/or medullary bone: flexible and fibrous bone matrix; transparent, hollow and pliable blood vessels; intravascular material, including in some cases, structures morphologically reminiscent of vertebrate red blood cells; and osteocytes with intracellular contents and flexible filipodia. The present study attempts to trace the occurrence of these four components in bone from specimens spanning multiple geological time periods and varied depositional environments. At least three of the four components persist in some skeletal elements of specimens dating to the Campanian. Fibrous bone matrix is more altered over time in morphology and less likely to persist than vessels and/or osteocytes. Vessels vary greatly in preservation, even within the same specimen, with some regions retaining pliability and other regions almost crystalline. Osteocytes also vary, with some retaining long filipodia and transparency, while others present with short and stubby filipodia and deeply pigmented nuclei, or are pigmented throughout with no nucleus visible. Alternative hypotheses are considered to explain the origin/source of observed materials. Finally, a two-part mechanism, involving first cross-linking of molecular components and subsequent mineralization, is proposed to explain the surprising presence of still-soft elements in fossil bone. These results suggest that present models of fossilization processes may be incomplete and that soft tissue elements may be more commonly preserved, even in older specimens, than previously thought. Additionally, in many cases, osteocytes with defined nuclei are preserved, and may represent an important source for informative molecular data.

Major cranial changes during Triceratops ontogeny.

This is the first cranial ontogenetic assessment of Triceratops, the well-known Late Cretaceous dinosaur distinguished by three horns and a massive parietal-squamosal frill. Our analysis is based on a growth series of 10 skulls, ranging from a 38 cm long baby skull to about 2 m long adult skulls. Four growth stages correspond to a suite of ontogenetic characters expressed in the postorbital horns, frill, nasal, epinasal horn and epoccipitals. Postorbital horns are straight stubs in early ontogeny, curve posteriorly in juveniles, straighten in subadults and recurve anteriorly in adults. The posterior margin of the baby frill is deeply scalloped. In early juveniles, the frill margin becomes ornamented by 17-19 delta-shaped epoccipitals. Epoccipitals are dorsoventrally compressed in subadults, strongly compressed and elongated in adults and ultimately merge onto the posterior frill margin in older adults. Ontogenetic trends within and between growth stages include: posterior frill margin transitions from scalloped to wavy and smooth; progressive exclusion of the supraoccipital from the foramen magnum; internal hollowing at the base of the postorbital horns; closure of the midline nasal suture; fusion of the epinasal onto the nasals; and epinasal expansion into a morphologically variable nasal horn. We hypothesize that the changes in horn orientation and epoccipital shape function to allow visual identity of juveniles, and signal their attainment of sexual maturity.

Fusion Patterns in the Skulls of Modern Archosaurs Reveal That Sutures Are Ambiguous Maturity Indicators for the Dinosauria.

The sutures of the skulls of vertebrates are generally open early in life and slowly close as maturity is attained. The assumption that all vertebrates follow this pattern of progressive sutural closure has been used to assess maturity in the fossil remains of non-avian dinosaurs. Here, we test this assumption in two members of the Extant Phylogenetic Bracket of the Dinosauria, the emu, Dromaius novaehollandiae and the American alligator, Alligator mississippiensis, by investigating the sequence and timing of sutural fusion in their skulls. As expected, almost all the sutures in the emu skull progressively close (i.e., they get narrower) and then obliterate during ontogeny. However, in the American alligator, only two sutures out of 36 obliterate completely and they do so during embryonic development. Surprisingly, as maturity progresses, many sutures of alligators become wider in large individuals compared to younger, smaller individuals. Histological and histomorphometric analyses on two sutures and one synchondrosis in an ontogenetic series of American alligator confirmed our morphological observations. This pattern of sutural widening might reflect feeding biomechanics and dietary changes through ontogeny. Our findings show that progressive sutural closure is not always observed in extant archosaurs, and therefore suggest that cranial sutural fusion is an ambiguous proxy for assessing maturity in non-avian dinosaurs.

Vertebral Adaptations to Large Body Size in Theropod Dinosaurs.

Rugose projections on the anterior and posterior aspects of vertebral neural spines appear throughout Amniota and result from the mineralization of the supraspinous and interspinous ligaments via metaplasia, the process of permanent tissue-type transformation. In mammals, this metaplasia is generally pathological or stress induced, but is a normal part of development in some clades of birds. Such structures, though phylogenetically sporadic, appear throughout the fossil record of non-avian theropod dinosaurs, yet their physiological and adaptive significance has remained unexamined. Here we show novel histologic and phylogenetic evidence that neural spine projections were a physiological response to biomechanical stress in large-bodied theropod species. Metaplastic projections also appear to vary between immature and mature individuals of the same species, with immature animals either lacking them or exhibiting smaller projections, supporting the hypothesis that these structures develop through ontogeny as a result of increasing bending stress subjected to the spinal column. Metaplastic mineralization of spinal ligaments would likely affect the flexibility of the spinal column, increasing passive support for body weight. A stiff spinal column would also provide biomechanical support for the primary hip flexors and, therefore, may have played a role in locomotor efficiency and mobility in large-bodied species. This new association of interspinal ligament metaplasia in Theropoda with large body size contributes additional insight to our understanding of the diverse biomechanical coping mechanisms developed throughout Dinosauria, and stresses the significance of phylogenetic methods when testing for biological trends, evolutionary or not.

A silicified bird from Quaternary hot spring deposits.

The first avian fossil recovered from high-temperature hot spring deposits is a three-dimensional external body mould of an American coot (Fulica americana) from Holocene sinters of Yellowstone National Park, Wyoming, USA. Silica encrustation of the carcass, feathers and colonizing microbial communities occurred within days of death and before substantial soft tissue degradation, allowing preservation of gross body morphology, which is usually lost under other fossilization regimes. We hypothesize that the increased rate and extent of opal-A deposition, facilitated by either passive or active microbial mediation following carcass colonization, is required for exceptional preservation of relatively large, fleshy carcasses or soft-bodied organisms by mineral precipitate mould formation. We suggest physico-chemical parameters conducive to similar preservation in other vertebrate specimens, plus distinctive sinter macrofabric markers of hot spring subenvironments where these parameters are met.

A New Brachylophosaurin Hadrosaur (Dinosauria: Ornithischia) with an Intermediate Nasal Crest from the Campanian Judith River Formation of Northcentral Montana.

BACKGROUND: Brachylophosaurini is a clade of hadrosaurine dinosaurs currently known from the Campanian (Late Cretaceous) of North America. Its members include: Acristavus gagslarsoni, which lacks a nasal crest; Brachylophosaurus canadensis, which possesses a flat paddle-shaped nasal crest projecting posteriorly over the dorsal skull roof; and Maiasaura peeblesorum, which possesses a dorsally-projecting nasofrontal crest. Acristavus, from the lower Two Medicine Formation of Montana (~81-80 Ma), is hypothesized to be the ancestral member of the clade. Brachylophosaurus specimens are from the middle Oldman Formation of Alberta and equivalent beds in the Judith River Formation of Montana; the upper Oldman Formation is dated 77.8 Ma. METHODOLOGY/PRINCIPAL FINDINGS: A new brachylophosaurin hadrosaur, Probrachylophosaurus bergei (gen. et sp. nov.) is described and phylogenetically analyzed based on the skull and postcranium of a large individual from the Judith River Formation of northcentral Montana (79.8-79.5 Ma); the horizon is equivalent to the lower Oldman Formation of Alberta. Cranial morphology of Probrachylophosaurus, most notably the nasal crest, is intermediate between Acristavus and Brachylophosaurus. In Brachylophosaurus, the nasal crest lengthens and flattens ontogenetically, covering the supratemporal fenestrae in large adults. The smaller nasal crest of Probrachylophosaurus is strongly triangular in cross section and only minimally overhangs the supratemporal fenestrae, similar to an ontogenetically earlier stage of Brachylophosaurus. Sutural fusion and tibial osteohistology reveal that the holotype of Probrachylophosaurus was relatively more mature than a similarly large Brachylophosaurus specimen; thus, Probrachylophosaurus is not simply an immature Brachylophosaurus. CONCLUSIONS/SIGNIFICANCE: The small triangular posteriorly oriented nasal crest of Probrachylophosaurus is proposed to represent a transitional nasal morphology between that of a non-crested ancestor such as Acristavus and the large flat posteriorly oriented nasal crest of adult Brachylophosaurus. Because Probrachylophosaurus is stratigraphically and morphologically intermediate between these taxa, Probrachylophosaurus is hypothesized to be an intermediate member of the Acristavus-Brachylophosaurus evolutionary lineage.

First Reported Cases of Biomechanically Adaptive Bone Modeling in Non-Avian Dinosaurs.

Predator confrontation or predator evasion frequently produces bone fractures in potential prey in the wild. Although there are reports of healed bone injuries and pathologies in non-avian dinosaurs, no previously published instances of biomechanically adaptive bone modeling exist. Two tibiae from an ontogenetic sample of fifty specimens of the herbivorous dinosaur Maiasaura peeblesorum (Ornithopoda: Hadrosaurinae) exhibit exostoses. We show that these outgrowths are cases of biomechanically adaptive periosteal bone modeling resulting from overstrain on the tibia after a fibula fracture. Histological and biomechanical results are congruent with predictions derived from this hypothesis. Histologically, the outgrowths are constituted by radial fibrolamellar periosteal bone tissue formed at very high growth rates, as expected in a process of rapid strain equilibration response. These outgrowths show greater compactness at the periphery, where tensile and compressive biomechanical constraints are higher. Moreover, these outgrowths increase the maximum bending strength in the direction of the stresses derived from locomotion. They are located on the antero-lateral side of the tibia, as expected in a presumably bipedal one year old individual, and in the posterior position of the tibia, as expected in a presumably quadrupedal individual at least four years of age. These results reinforce myological evidence suggesting that Maiasaura underwent an ontogenetic shift from the primitive ornithischian bipedal condition when young to a derived quadrupedal posture when older.

Age and growth dynamics of Tyrannosaurus rex.

Tyrannosaurus rex is the most commonly found North American latest Cretaceous theropod, but until the 1980s only five specimens had been discovered, and no more than six have received a full description. Consequently there has been little information on how old Tyrannosaurus specimens were at maturity or death. Histological analysis of seven individuals provided, for the first time, an opportunity to assess the age represented by the bone cortex, to estimate the average individual age of these skeletons, to determine whether they represented fully grown individuals, and to predict their individual longevity. Though a range of ages (15-25 years) was found for the specimens studied, the seven individuals demonstrate that T. rex reached effectively full size in less than 20 years. The growth rate of T. rex was comparable to that of the African elephant, which has a similar mass and time to maturity. Some of the known specimens of T. rex did not quite reach full size; others do not seem to have survived long after achieving it.

Remarkable preservation of undigested muscle tissue within a Late Cretaceous tyrannosaurid coprolite from Alberta, Canada.

Exceptionally detailed soft tissues have been identified within the fossilized feces of a large Cretaceous tyrannosaurid. Microscopic cord-like structures in the coprolitic ground mass are visible in thin section and with scanning electron microscopy. The morphology, organization, and context of these structures indicate that they are the fossilized remains of undigested muscle tissue. This unusual discovery indicates specific digestive and taphonomic conditions, including a relatively short gut-residence time, rapid lithification, and minimal diagenetic recrystallization. Rapid burial of the feces probably was facilitated by a flood event on the ancient coastal lowland plain on which the fecal mass was deposited.