Schema-based learning of adaptable and flexible prey-catching in anurans I. The basic architecture

A motor action often involves the coordination of several motor synergies and requires flexible adjustment of the ongoing execution based on feedback signals. To elucidate the neural mechanisms underlying the construction and selection of motor synergies, we study prey-capture in anurans. Experimental data demonstrate the intricate interaction between different motor synergies, including the interplay of their afferent feedback signals (Weerasuriya 1991; Anderson and Nishikawa 1996). Such data provide insights for the general issues concerning two-way information flow between sensory centers, motor circuits and periphery in motor coordination. We show how different afferent feedback signals about the status of the different components of the motor apparatus play a critical role in motor control as well as in learning. This paper, along with its companion paper, extend the model by Liaw et al. (1994) by integrating a number of different motor pattern generators, different types of afferent feedback, as well as the corresponding control structure within an adaptive framework we call Schema-Based Learning. We develop a model of the different MPGs involved in prey-catching as a vehicle to investigate the following questions: What are the characteristic features of the activity of a single muscle? How can these features be controlled by the premotor circuit? What are the strategies employed to generate and synchronize motor synergies? What is the role of afferent feedback in shaping the activity of a MPG? How can several MPGs share the same underlying circuitry and yet give rise to different motor patterns under different input conditions? In the companion paper we also extend the model by incorporating learning components that give rise to more flexible, adaptable and robust behaviors. To show these aspects we incorporate studies on experiments on lesions and the learning processes that allow the animal to recover its proper functioning     

A new pipoid frog (Anura,Pipimorpha) from the Paleogene of Patagonia. Paleobiogeographical implications

The aim of the present contribution is to describe a new genus and species of Pipoidea from the Huitrera Formation (Eocene) from Patagonia, Argentina. The new genus shows a unique combination of characters indicating that it is a valid taxon different from other pipimorphs, including the coeval Llankibatrachus truebae. The phylogenetic analysis resulted in the nesting of the new taxon within a previously unrecognized endemic clade of South American aglossans. This new clade turns out to be the sister-group of crown-group Pipidae. This phylogenetic proposal reinforces the hypothesis sustaining the dispersal of pipids between Africa and South America through an island chain or a continental bridge across the Atlantic Ocean by Early Tertiary times.     

New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis

Retinal lesions or other ocular manifestations are serious consequences of infection with the protozoan parasite Toxoplasma gondii. Whilst classically considered a consequence of congenital transmission, recent screening studies estimated that 2% of T. gondii seropositive persons in Europe and North America have retinal lesions, most of them persisting unnoticed. The situation is more dramatic in South America, probably due to the predominance of virulent strains. Some of these strains seem to exhibit ocular or neuronal tropism and are responsible for severe ocular lesions. Despite the medical importance, the physiopathological mechanisms have only recently begun to be elucidated. The particular immune-privileged situation in the eye has to be considered. Studies on French patients showed low or undetectable ocular parasite loads, but a clear Th1/Th17 type immune reaction. Suitable mouse models have appeared in the last few years. Using such a model, IL-17A proved to impair parasite control and induce pathology. In contrast, in South American patients, the parasite seems to be much less efficiently controlled through a Th2 type or suppressive immune response that favors parasite replication. Finally, several host genetic markers controlling immune response factors have been associated with ocular involvement of T. gondii infection, mainly in South America.     

Première indication de la présence de phorusracidés,famille d'oiseaux géants d'amérique du sud,dans le tertiaire Européen: Ameghinornis nov. gen. (Aves,ralliformes) des phosphorites du quercy,France

Some bird remains, from the «Phosphorites du Quercy deposits are closely related to the corresponding bones of Phorusrhacidae, an extinct family of birds which underwent an extensive diversification in South America from Oligocene to Pleistocene times. It included many giant flightless species. The form of the Quercy is a rather primitive one, of medium size and of probably somewhat reduced flying ability.The simultaneous occurrence of Phorusrhacidae inFrance and South America is of great biogeographical significance. The Phorusrhacidae seem most probably to be the descendants of a rather primitive group of Ralliformes, already diversified in Gondwanaland prior to its separation into different continental masses.     

Mitochondrial DNA and the peopling of South America

The initial peopling of South America is largely unresolved, in part because of the unique distribution of genetic diversity in native South Americans. On average, genetic diversity estimated within Andean populations is higher than that estimated within Amazonian populations. Yet there is less genetic differentiation estimated among Andean populations than estimated among Amazonian populations. One hypothesis is that this pattern is a product of independent migrations of genetically differentiated people into South America. A competing hypothesis is that there was a single migration followed by regional isolation. In this study we address these hypotheses using mtDNA hypervariable region 1 sequences representing 21 South American groups and include new data sets for four native Peruvian communities from Tupe, Yungay, and Puno. An analysis of variance that compared the combined data from western South America to the combined data from eastern South America determined that these two regional data sets are not significantly different. As a result, a migration from a single source population into South America serves as the simplest explanation of the data.     

Records and Stratigraphical Ranges of South American Tayassuidae (Mammalia, Artiodactyla)

Tayassuidae represent one of the first mammalian immigrants that entered South America during the “Great American Biotic Interchange.” However, the exact moment of its arrival for the first time in South America is controversial. Three genera are recognized in South America: Platygonus, Catagonus, and Tayassu. This paper aims to: (1) review the paleontological record of the South American Tayassuidae and update it; and (2) discuss its geographical and statigraphical distribution pattern in South America. The genus Platygonus (middle Pliocene to early Pleistocene) is registered in Argentina, Uruguay, Colombia, and Bolivia; Catagonus (late Pliocene? to Recent) in Argentina, Uruguay, Brazil, and Bolivia; and Tayassu (middle Pleistocene to Recent) in Argentina, Uruguay, and Brazil. Platygonus and Catagonus have adaptations to dry and relatively open environments; in contrast, Tayassu is adapted mainly to humid climates and woodland and forest environments. The faunal changes that took place since the middle-late Pliocene could have been strongly influenced by climate. Open and arid environments developed during the glacial cycles, allowing the latitudinal expansion of Platygonus and Catagonus. Considering ecological and anatomical information, it is possible to infer that Platygonus species were replaced by those of Catagonus since the middle Pleistocene, probably due to a reduction of the open environments to which Platygonus species were better adapted. The alternation of these mainly arid or semiarid, cold conditions with warmer and more humid short pulses would have allowed the posterior expansion of Tayassu species. According to phylogenetic analysis and chronological as well as geographical evidence, Platygonus and Catagonus represent two Tayassuidae lineages that originated in North America and then migrated to South America. This migration would have occurred on more than one occasion and with different taxa. Evidence indicates that Tayassu represents a lineage that differentiated in the Southern Hemisphere and then migrated to North America.     

Genetic Diversity of Neotropical Myotis (Chiroptera: Vespertilionidae) with an Emphasis on South American Species

Background

Cryptic morphological variation in the Chiropteran genus Myotis limits the understanding of species boundaries and species richness within the genus. Several authors have suggested that it is likely there are unrecognized species-level lineages of Myotis in the Neotropics. This study provides an assessment of the diversity in New World Myotis by analyzing cytochrome-b gene variation from an expansive sample ranging throughout North, Central, and South America. We provide baseline genetic data for researchers investigating phylogeographic and phylogenetic patterns of Myotis in these regions, with an emphasis on South America.

Methodology and Principal Findings

Cytochrome-b sequences were generated and phylogenetically analyzed from 215 specimens, providing DNA sequence data for the most species of New World Myotis to date. Based on genetic data in our sample, and on comparisons with available DNA sequence data from GenBank, we estimate the number of species-level genetic lineages in South America alone to be at least 18, rather than the 15 species currently recognized.

Conclusions

Our findings provide evidence that the perception of lower species richness in South American Myotis is largely due to a combination of cryptic morphological variation and insufficient sampling coverage in genetic-based systematic studies. A more accurate assessment of the level of diversity and species richness in New World Myotis is not only helpful for delimiting species boundaries, but also for understanding evolutionary processes within this globally distributed bat genus.     

Mitochondrial DNA based phylogeny of the woodpecker genera Colaptes and Piculus,and implications for the history of woodpecker diversification in South America

The woodpecker genus Colaptes (flickers) has its highest diversity in South America and the closely related genus Piculus is restricted to South and Central America. Two species of flickers occur in North America, and one species is endemic to Cuba. We conducted a Bayesian phylogenetic analysis of three mitochondrial encoded genes (cyt b, COI, 12S rRNA) and confirmed that the two genera are paraphyletic. Three species historically classified as Piculus are actually flickers. We found that the Cuban endemic C. fernandinae is the most basal species within the flickers and that the Northern Flicker is the next most basal species within the Colaptes lineage. The South American clade is most derived. The age of the South American diversification is estimated to be 3.6 MY, which is synchronous with the emergence of the Isthmus of Panama. The pattern of diversification of South American flickers is common among South American woodpeckers. Although woodpeckers have their greatest diversity in South America, we hypothesize that woodpeckers (Family Picidae) originated in Eurasia, dispersed to North America via the Bering land bridge, and multiple lineages entered South America as the Isthmus approached its final closing.     

The exploitation of megafauna during the earliest peopling of the Americas: An examination of nineteenth-century fossil collections

This paper describes human-modified bones originally from the Pampas region, and that form part of nineteenth-century fossil collections of native fauna. We describe the morphological and configurational features of the marks, relate them to the various stages in the butchering process, and discuss access type. An examination of various different American sites is used to interpret this evidence at a coarse-grained level. Although these collections are more biased than current archaeological materials with regard to their sedimentary origin and previous handling, the application of modern technology has allowed us to obtain new data. Therefore, despite their complex history, these artefacts can be incorporated into the broader body of modern archaeological research. This type of study adds new value to our historic heritage and underscores its usefulness in modern enquiries, in this case, related to the topic of how Homo sapiens interacted with the native fauna in the southern Cone of South America.     

Dispersal spectra,diaspore size and the importance of endozoochory in the equatorial Andean montane forests

The dispersal syndrome hypothesis states that plant diaspores show morphological features that are the results of adaptation for dispersal by a particular vector. This can enable to identify the relative importance of dispersal agents within plant communities. Nevertheless, there is still little information about seed dispersal spectra and diaspore traits related to different dispersal agents in the equatorial montane forests, despite their high biodiversity and important ecosystem services as watersheds for human communities. Due to an increase in environmental stress at high elevations a reduction in the prevalence of endozoochory, and a reduction in the size of endozoochorous diaspores in plant assemblages could be expected. We reviewed published data from 64 Andean cloud forest plots to assess the dispersal spectra, the incidence of different traits related to seed dispersal, and the distribution of dispersal syndromes within cloud forests of northern South America. We then evaluated two questions related to seed dispersal in these forests: (1) Does the number and percentage of endozoochorous species in woody plant assemblages decrease at higher elevation? and (2) Does the mean diaspore size of endozoochorously dispersed tree assemblages decrease with elevation?     

Chromosome studies in southern species of Mimosa (Fabaceae,Mimosoideae) and their taxonomic and evolutionary inferences

In this work, chromosome numbers and karyotype parameters of 36 taxa of the genus Mimosa were studied, especially from the southern South America center of diversification. Results support that x = 13 is the basic chromosome number in the genus. Polyploidy is very frequent, ca. 56 % of the total of the studied species here are polyploid, confirming that polyploids are more frequent at higher latitudes. The most common ploidy levels found are 2x and 4x, but some species studied exhibit 6x and 8x. In different groups, several ploidy levels were found. Parameters of chromosome size show statistically significant differences between close species, and asymmetry index A ₂ exhibited low variation between them. It is possible to infer variations of chromosome size between diploids and tetraploids and between basal and derived taxa. The present studies confirm or reveal polyploidy in several groups of South America which are highly diversified in the southernmost area of distribution of the genus, such as sect. Batocaulon ser. Stipellares and sect. Calothamnos. Our data are discussed in a taxonomic context, making inferences about the origin of some polyploid taxa. Polyploidy could be an important phenomenon that increases the morphologic diversity and specific richness in southern South America. On basis of our data, it is possible to hypothesize hybridization between same-ploidy level or different ploidy level taxa. As already shown in the literature, our results confirm the importance of the polyploidy in the speciation of the genus.     

“South American” Marsupials from the Late Cretaceous of North America and the Origin of Marsupial Cohorts

Newly described marsupial specimens of Judithian (late Campanian) and Lancian (Maastrichtian) age in the western interior of North America (Wyoming to Alberta) have dental morphologies consistent with those expected in comparably aged sediments in South America (yet to be found). Three new Lancian species are referable to the didelphimorphian Herpetotheriidae, which suggests that the ameridelphian radiation was well under way by this time. The presence of a polydolopimorphian from Lancian deposits with a relatively plesiomorphic dental morphology and an additional polydolopimorphian taxon from Judithian deposits with a more derived molar form indicate that this lineage of typically South American marsupials was diversifying in the Late Cretaceous of North America. This study indicates that typical South American lineages (e.g. didelphimorphians and polydolopimorphians) are not the result of North American peradectian progenitors dispersing into South America at the end of the Cretaceous (Lancian), or at the beginning of the Paleocene (Puercan), and giving rise to the ameridelphian marsupials. Instead, these lineages, and predictably others as well, had their origins in North America (probably in more southerly latitudes) and then dispersed into South America by the end of the Cretaceous. Geophysical evidence concerning the connections between North and South America in the Late Cretaceous is summarized as to the potential for overland mammalian dispersal between these places at those times. Paleoclimatic reconstructions are considered, as is the dispersal history of hadrosaurine dinosaurs and boid snakes, as to their contribution to an appraisal of mammalian dispersals in the Late Cretaceous. In addition, we present a revision of the South American component of the Marsupialia. One major outcome of this process is that the Polydolopimorphia is placed as Supercohort Marsupialia incertae sedis because no characteristics currently known from this clade securely place it within one of the three named marsupial cohorts.     

The evolution of the Brachycytheride Ostracoda in the context of the breakup of Pangea

The ostracode Family Brachycytheridae is one of the most common taxa of Late Cretaceous shallow marine microfossils, and occurs in the marginal basins of Africa, Madagascar, the Middle East, South and North America and the Caribbean region. Their migration pathways were controlled largely by the plate tectonic events associated with the breakup of Pangea and the formation of the North and South Atlantic Oceans, the Caribbean Sea and the Gulf of Mexico. This study is an attempt to combine specific information on the plate tectonic events as evidenced by the timings of continental-scale structural movements, sea level dynamics (largely the product of mantle processes), the creation and destruction of migration pathways, and taxonomy of the group, and thus relate deep-seated Earth processes, surficial expression of these movements, and biological evolution. Although the Late Cretaceous Brachycytheridae are very widely distributed, their use for correlation has been hampered due to their conservative external morphology, with all of them having a posteriorly-sloped dorsal margin and a swollen ventrolateral carapace, and all of them were long assigned to a single genus, Brachycythere. In 2002, Puckett defined a new genus, Acuminobrachycythere, based on a distinctive clade of North American brachycytherids. It was also recognized at that time that the members of the family from Gondwana (mainly Africa and South America) were distinct in having a single (unsplit) second adductor muscle scar, whereas all of those from North America had a split second adductor. This tiny difference holds significant paleogeographic—and presumably evolutionary—information. Two new genera are therefore described herein, Sapucariella, which is restricted to Gondwana, and Tricostabrachycythere, which is an early, short-lived genus that occurred around the margins of Gondwana. Three new species are described and one is re-described. In all, there are six genera in the Family Brachycytheridae, which include Acuminobrachycythere, Brachycythere, Kaesleria (which is restricted to the Middle East), Opimocythere (which evolved in the Paleogene of North America and is the last of the Brachycytheridae), Sapucariella and Tricostabrachycythere. Taxonomic information is presented for 85 species, including new images of many of the type specimens. A summary of the breakup of Pangea is also presented, with precise paleogeographic reconstructions based on global magnetic anomalies.     

New Glyptodont from the Codore Formation (Pliocene), Falcón State, Venezuela, its relationship with theAsterostemma problem, and the paleobiogeography of the Glyptodontinae

One of the basal Glyptodontidae groups is represented by the Propalaehoplophorinae (late Oligocene — middle Miocene), whose genera (Propalaehoplophorus, Eucinepeltus, Metopotoxus, Cochlops, andAsterostemma) were initially recognized in Argentinian Patagonia. Among these,Asterostemma was characterized by its wide latitudinal distribution, ranging from southernmost (Patagonia) to northernmost (Colombia, Venezuela) South America. However, the generic assignation of the Miocene species from Colombia and Venezuela (A.? acostae, A. gigantea, andA. venezolensis) was contested by some authors, who explicitly accepted the possibility that these species could correspond to a new genus, different from those recognized in southern areas. A new comparative study of taxa from Argentinian Patagonia, Colombia and Venezuela (together with the recognition of a new genus and species for the Pliocene of the latter country) indicates that the species in northern South America are not Propalaehoplophorinae, but represent the first stages in the cladogenesis of the Glyptodontinae glyptodontids, the history of which was heretofore restricted to the late Miocene — early Holocene of southernmost South America. Accordingly, we propose the recognition of the new genusBoreostemma for the species from northern South America and the restriction ofAsterostemma to the Miocene of Patagonia. Thus, the available data indicate that the Glyptodontinae would in fact have arisen in the northernmost regions of this continent. Their arrival to more southerly areas coincides with the acme of the “Age of Southern Plains”. The Propalaehoplophorinae are geographically restricted to Patagonia.     

Further evidence for the presence of holoptychiid porolepiforms (Sarcopterygii,Dipnomorpha) from the Frasnian of Colombia

The Devonian (Frasnian) of Colombia has provided important information on the taxonomical diversity and palaeobiogeographic relationships of the Late Devonian marginal marine vertebrate faunas of South America (northwestern margin of Gondwana). This fauna is mostly composed of Gondwanan endemics, but includes two taxa also known in Euramerica: the antiarchan placoderm Asterolepis and the porolepiform sarcopterygian Holoptychius. The occurrence of holoptychiid porolepiforms in Colombia was previously suggested based solely on the presence of scales attributed to Holoptychius, although with caution. Here, we describe further holoptychiid remains that include a large isolated tooth with dendrodont microstructure, typical of porolepiforms, and additional Holoptychius scales. These new findings increase the record of holoptychiid porolepiform occurrences in Gondwana and suggest that dispersion of fish faunas between Euramerica and Gondwana by the beginning of the Late Devonian was possible through South America.     

Geographic variation in a widespread neotropical species,Mouriri myrtilloides (Melastomataceae)

Geographic variation inMouriri myrtilloides (Swartz) Poiret is analyzed on a morphological basis. Plants from Central America have medium puberulence, long calyx lobes, and short petioles and pedicels; those from Jamaica and Haiti are glabrous and have short calyx lobes, petioles, and pedicels; while those from Cuba have maximum puberulence, medium calyx lobes and pedicels, and long petioles. Glabrous or near-glabrous plants also appear rarely and more or less at random in Central and South America. Distribution in South America is limited to the northwest quarter of the continent. Most plants of this region are similar to those of Central America except that they have slightly shorter calyx lobes. However, the very short calyx lobes of plants from Jamaica and Haiti often occur in South America and rarely in Panama. Furthermore, most features of the Cuban plants appear in those of the Orinoco Basin, while individuals with one of these features, strong puberulence, are occasional elsewhere in South America. It is suggested that the complex originated in northwest South America and diversified there, subsequently radiating north and west. Considerations and problems related to such a dispersal are discussed. Taxonomically the complex is treated as consisting of four subspecies: ssp.parvifolia of Central America and much of the north-west quarter of South America, ssp.myrtilloides of Jamaica and southwest Haiti, ssp.acuta of Cuba, and ssp.orinocensis of the Orinoco Basin, the latter being a new taxon.     

Molecular phylogeny and evolutionary history of the tit-tyrants (Aves: Tyrannidae)

Tit-tyrants of the genus Anairetes presently consist of six species; five inhabit various regions along the Andean cordillera of South America and one is endemic to the Juan Fernandez Islands off the coast of Chile. Data from mtDNA ND2 and Cyt b sequences were used to construct a phylogeny for all Anairetes species as well as Uromyias agilis, a closely related genus, and Stigmatura as an outgroup, to determine their relationships and history of radiation in South America. Results strongly supported the following paired relationships: A. nigrocristatus-A. reguloides, A. flavirostris-A. alpinus, and A. parulus-A. fernandezianus. This dataset, however, could not resolve basal nodes; therefore relationships among these pairs remains obscure. Moreover the genus Uromyias, controversially separated on morphological criteria from Anairetes, fell within the Anairetes clade, although its exact position could not be ascertained with confidence. The molecular data indicate that this group probably radiated within the past 2 million years, concomitant with highly accentuated cycles of global climatic change. Certain high altitude areas within the Andes may have been stable during global climatic changes and may have served as refugia during the Plio-Pleistocene.     

Modeling potential invasion of stored-product pest Cryptamorpha desjardinsii (Guérin-Méneville, 1844) (Coleoptera: Silvanidae) with emphasis on newly recorded areas

Desjardin’s flat bark beetle – Cryptamorpha desjardinsii (Guérin-Méneville, 1844) – is a widespread invasive species that can become a stored products pest and threaten native species. Our objectives were to provide the first detailed records of this species to South America and New Caledonia, and model suitable areas of C. desjardinsii globally based on different Ecological Niche Models (ENM). We combine several techniques to predict the ecological niche of C. desjardinsii using distribution records and a set of environmental predictor variables. Overall, the species appears to be well established in tropical and subtropical regions, particularly in islands, with new and previous records predicted by the consensus model. In South America, the driest and coldest regions were predicted to be less suitable for the occurrence of C. desjardinsii. In the current global spread context, these findings are pivotal to predicting areas of high risk of invasion of C. desjardinsii.     

Karyotype and AFLP data reveal the phylogenetic position of the Brazilian endemic Hypochaeris catharinensis (Asteraceae)

The genus Hypochaeris offers an excellent model for studies of recent adaptive radiation in the South American continent. We used karyotype analysis with chromomycin?A₃ (CMA₃)/4??,6-diamidino-2-phenylindole (DAPI) banding and fluorescence in?situ hybridization (FISH), and amplified fragment length polymorphism (AFLP) fingerprinting to investigate for the first time the Brazilian endemic H.?catharinensis and define its position within the South American group of species. Strong CMA-positive signals were seen at the end of both arms of chromosome?3 and at the end of the long arm of chromosome?4. DAPI bands were only detected in subterminal position on short arm of chromosome?4. FISH with 5S and 35S ribosomal DNA (rDNA) probes revealed a single 5S rDNA locus on short arm of chromosome?2, typical for all other South American Hypochaeris taxa analyzed to date. The 35S rDNA locus was identified at subterminal position on the short arm of chromosome?3, as reported so far for only two of the known species (H.?lutea and H.?patagonica). The AFLP study included 55 individuals, comprising nine species of the South American Hypochaeris plus their putative ancestor H.?angustifolia. Eleven AFLP primer combinations generated a total of 401 fragments, of which 388 (96.7%) were polymorphic. High genetic similarities were observed among taxa, with all South American Hypochaeris species falling into one main cluster [100% bootstrap (BS)]. Hypochaeris catharinensis is closely related to H.?lutea (82% BS), forming a well-separated subcluster within the South American species. Taken together, the karyological and AFLP data contribute to the placement of H.?catharinensis within the phylogenetic framework of South American species of Hypochaeris and allow the definition of a novel and well-resolved phylogenetic group (the Lutea group).     

Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus vulgaris L.) in the Americas

The wild common bean (Phaseolus vulgaris) is widely but discontinuously distributed from northern Mexico to northern Argentina on both sides of the Isthmus of Panama. Little is known on how the species has reached its current disjunct distribution. In this research, chloroplast DNA polymorphisms in seven non-coding regions were used to study the history of migration of wild P. vulgaris between Mesoamerica and South America. A penalized likelihood analysis was applied to previously published Leguminosae ITS data to estimate divergence times between P. vulgaris and its sister taxa from Mesoamerica, and divergence times of populations within P. vulgaris. Fourteen chloroplast haplotypes were identified by PCR-RFLP and their geographical associations were studied by means of a Nested Clade Analysis and Mantel Tests. The results suggest that the haplotypes are not randomly distributed but occupy discrete parts of the geographic range of the species. The current distribution of haplotypes may be explained by isolation by distance and by at least two migration events between Mesoamerica and South America: one from Mesoamerica to South America and another one from northern South America to Mesoamerica. Age estimates place the divergence of P. vulgaris from its sister taxa from Mesoamerica at or before 1.3 Ma, and divergence of populations from Ecuador-northern Peru at or before 0.6 Ma. As these ages are taken as minimum divergence times, the influence of past events, such as the closure of the Isthmus of Panama and the final uplift of the Andes, on the migration history and population structure of this species cannot be disregarded.