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Carboxyl ester lipase (CEL) is a digestive enzyme which is secreted by acinar cells in the pancreas and released into the intestines. In addition to the globular domain responsible for lipase activity, CEL contains a C-terminal region encoded by a Variable Number of Tandem Repeats (VNTR) sequence. Each repeat consists of 11 amino acid residues. The function of the VNTR region is enigmatic, but we know that it is disordered, becomes O-glycosylated and is necessary for proper secretion of CEL. The CEL VNTR is also highly polymorphic and at least two variants are confirmed to be pathogenic: CEL-MODY and CEL-HYB. These variants are associated with cellular aggregation and pancreatic disease. A third variant with only three VNTR repeats, CEL-3R, has been discovered in a Danish family, in which it co-segregates with diabetes. However, we do not know if CEL-3R is pathogenic. In this study, we attempted to investigate the properties of the CEL VNTR with a special emphasis on the Danish CEL-3R variant. To examine if CEL-3R is pathogenic, we expressed it in HEK293 cells and compared its cellular localisation and secretion to the normal CEL protein and to the pathogenic CEL-HYB variant. Further, to better understand the role of the VNTR in general, we examined its evolution and characteristics in vertebrate species by performing a phylogenetic analysis. Finally, we predicted the conformations of the CEL-3R VNTR by molecular simulations. We found that the CEL-3R variants exhibited levels of aggregation in HEK293 cells comparable to the pathological CEL-HYB variant. Still, CEL-3R displayed increased secretion compared to CEL-HYB. Moreover, we found that the CEL VNTR was present only in mammalian species. The mammalian VNTR sequences were relatively conserved, but exhibited a diverse number of repeats. A short post-VNTR sequence was also well conserved among mammals of the clade Eutheria. We were not able to correlate the number of VNTR repeats with any specific traits of the investigated mammalian species. The molecular simulations of the VNTR region of CEL-3R predicted that it forms unstructured coils and temporary turns, β-bridges and β-sheets. The O-glycosylated VNTR structure displayed a similar pattern. In conclusion, although more transfection experiments are needed, we found that the Danish CEL-3R variant may show signs of pathological aggregation. We also confirmed that the CEL VNTR probably evolved sometime between the origin of Synapsida and the formation of Eutheria and Metatheria. Finally, the typical ’EATPVPPTGDS’ repeats in the VNTR may be important for the formation of secondary structures that induce the VNTR to become compact, but still disordered. Karboksylester-lipase (CEL) er eit fordøyingsenzym som vert skilt ut i tarmen frå acinærcellene i bukspyttkjertelen. I tillegg til det globulære domenet som er ansvarleg for lipase-aktiviteten, inneheld CEL ein C-terminal region koda av ein sekvens med eit variabelt tal av tandemrepetisjonar, ein såkalla VNTR region. Kvar repetisjon kodar for 11 aminosyrer. Funksjonen til VNTR-regionen er ikkje kjent, men me veit at han er uordna, blir O-glykosylert og er naudsynt for effektiv utskiljing av CEL. VNTR-regionen er også særs variabel (polymorf) og minst to variantar er stadfesta å vera patogene: CEL-MODY og CEL-HYB. Desse variantane er knytt til cellulær aggregering og sjukdom i bukspyttkjertelen. Ein tredje variant med berre tre repetisjonar, CEL-3R, har vorte oppdaga i ein dansk familie der han finst hjå dei medlemmane som har diabetes. Likevel veit me ikkje om CEL-3R er patogen. I denne studia utforska me eigenskapane til VNTR-regionen til CEL, med særleg merksemd på den danske CEL-3R-varianten. For å undersøkja om CEL-3R er patogen, uttrykte me han i HEK293-celler og samanlikna distribusjon og utskiljing mot det normale CEL-proteinet i tillegg til den patogene CEL-HYB-varianten. Deretter, for å forstå rolla til VNTR-regionen, undersøkte me korleis han kunne vorte utvikla gjennom evolusjonen. Til slutt prøvde me ved å nytte simulasjonar å seie noko om kva konformasjonar VNTR-regionen til CEL-3R kan ha. Me fann at CEL-3R-variantar viste eit nivå av aggregering i HEK293-celler som kunne minna om den patologiske CEL-HYB-varianten. Likevel hadde CEL-3R auka utskiljing samanlikna med CEL-HYB. Vidare fann me at VNTR-regionen berre var til stades i pattedyrartar. VNTR-sekvensane var konserverte i pattedyr, men talet på repetisjonar varierte sterkt. Ein kort post-VNTR-sekvens var godt konservert i kladen Eutheria (placentale pattedyr). Me fann ingen samanheng mellom talet på repetisjonar i VNTR-regionen og eigenskapar til dyra som vart undersøkte. Dei molekylære simulasjonane av VNTR-regionen til CEL-3R predikerte at han kan danna ustrukturerte kveilar og kortvarige vendingar, β-bruer og β-flak. Me konkluderte med at den danske CEL-3R-varianten viser teikn på patologisk aggregering, men at me må gjera fleire eksperiment for å få stadfesta dette. Me fann også at VNTR-regionen til CEL sannsynlegvis vart utvikla ein gong mellom fyrste førekomst av kladen Synapsida (pattedyrliknande krypdyr) og forgreininga til Eutheria og Metatheria (pungdyr). Til slutt fann me at den typiske ’EATPVPPTGDS’-repetisjonen i VNTR-regionen kan vera viktig for danninga av sekundære strukturar. Desse strukturane fører til at regionen vert kompakt, men likevel uordna. Masteroppgave i molekylærbiologi MOL399 MAMN-MOL

Background Different types of proteins diverge at vastly different rates. Moreover, the same type of protein has been observed to evolve with different rates in different phylogenetic lineages. In the present study we measured the rates of protein evolution in Eutheria (placental mammals) and Metatheria (marsupials) on a genome-wide basis and we propose that the gene position in the genome landscape has an important influence on the rate of protein divergence. Results We analyzed a protein-encoding gene set (n = 15,727) common to 16 mammals (12 Eutheria and 4 Metatheria). Using sliding windows that averaged regional effects of protein divergence we constructed landscapes in which strong and lineage-specific regional effects were seen on the molecular clock rate of protein divergence. Within each lineage, the relatively high rates were preferentially found in subtelomeric chromosomal regions. Such regions were observed to contain important and well-studied loci for fetal growth, uterine function and the generation of diversity in the adaptive repertoire of immunoglobulins. Conclusions A genome landscape approach visualizes lineage-specific regional differences between Eutherian and Metatherian rates of protein evolution. This phenomenon of chromosomal position is a new element that explains at least part of the lineage-specific effects and differences between proteins on the molecular clock rates.

We have made quantitative volumetric analyses of cerebral cortical (pallial) structures in the brains of three species of monotreme (Ornithorhynchus anatinus, Tachyglossus aculeatus, Zaglossus bruijni) and compared the findings with similar measurements in a range of therian mammals (6 marsupials and 50 placentals). We have found that although the iso- and periallocortical grey matter volume of the monotremes is about what would be expected for their brain size, the proportion of iso- and periallocortical white matter in monotremes is substantially lower than that in the forebrains of therians. This suggests that the forebrains of the three monotremes have fewer association, commissural and/or projection connections than those of similarly sized forebrains of therian mammals. We also found that the iso- and periallocortex of the platypus is relatively smooth-surfaced compared to similarly sized brains of therian mammals, with a distinct caudal shift in the positioning of cortical white matter in the forebrain, consistent with expansion of the posterior thalamic radiation. Central laminated olfactory structures (anterior olfactory nucleus and piriform cortex) are large in the tachyglossid monotremes (Tachyglossus aculeatus and Zaglossus bruijni) and large in xenarthran placental mammals, suggesting convergence of the forebrain structure of monotreme formivores with that of similarly specialized therians like the xenarthrans Myrmecophaga tridactyla and Dasypus novemcinctus. Fil: Ashwell, Ken. University of New South Wales; Australia Fil: Gurovich, Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigación Esquel de Montaña y Estepa Patagóica. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Esquel. Centro de Investigación Esquel de Montaña y Estepa Patagónica; Argentina. University of South Wales; Australia

Background Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Also related, but not as closely, is B. besnoiti, the cause of bovine besnoitiosis. Methods Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. Results One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti and Neospora caninum, were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. Conclusions Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts. Graphical Abstract

Molecular estimates of the divergence of placental and marsupial mammals and their broader clades (Eutheria and Metatheria, respectively) fall primarily in the Jurassic period. Supporting these estimates, Juramaia-the oldest purported eutherian-is from the early Late Jurassic (160 million years ago) of northeastern China. Sinodelphys-the oldest purported metatherian-is from the same geographic area but is 35 million years younger, from the Jehol biota. Here we report a new Jehol eutherian, Ambolestes zhoui, with a nearly complete skeleton that preserves anatomical details that are unknown from contemporaneous mammals, including the ectotympanic and hyoid apparatus. This new fossil demonstrates that Sinodelphys is a eutherian, and that postcranial differences between Sinodelphys and the Jehol eutherian Eomaia-previously thought to indicate separate invasions of a scansorial niche by eutherians and metatherians-are instead variations among the early members of the placental lineage. The oldest known metatherians are now not from eastern Asia but are 110 million years old from western North America, which produces a 50-million-year ghost lineage for Metatheria.

A state-of-the-art review of the Cenozoic fossil record from Western Amazonia is provided, based on literature and new data (regarding Paleogene native ungulates). It allows summarizing the evolution and dynamics of middle Eocene–Holocene mammalian guilds, at the level of species, families, and orders. Major gaps in the Western Amazonian mammal record occur in the pre-Lutetian and early Miocene intervals, and in the Pliocene epoch. Twenty-three orders, 89 families, and 320 species are recognized in the fossil record, widely dominated by eutherians from the middle Eocene onward. Probable Allotheria (Gondwanatheria) occur only in the earliest interval, whereas Metatheria and Eutheria are conspicuous components of any assemblage. Taxonomic diversity was probably fairly constant at the ordinal level (~12–14 orders in each time slice considered) and much more variable in terms of family and species richness: if most intervals are characterized by 40–50 co-occurring species and 19–31 co-occurring families, the early Miocene period illustrates a depauperate fauna (21 species, 17 families), strongly contrasting with the late Miocene climactic guild (82 species, 38 families). Recent mammalian taxonomic diversity from Western Amazonia (12 orders, 37 families, and 286 species) is at odds with all past intervals, as it encompasses only three orders of South American origin (Didelphimorphia, Cingulata, and Pilosa) but four North American immigrant orders (Artiodactyla, Perissodactyla, Carnivora, and Lagomorpha). In terms of taxonomic diversity, recent mammalian guilds are fully dominated by small-sized taxa (Chiroptera, Rodentia, and Primates). This overview also confirms the scarcity of large mammalian flesh-eaters in ancient Neotropical mammalian assemblages. The pattern and the timing of mammalian dispersals from northern landmasses into Western Amazonia are not elucidated yet. Fil: Antoine, Pierre Olivier. Université de Montpellier; Francia Fil: Salas Gismondi, Rodolfo. Université de Montpellier; Francia. Universidad Nacional Mayor de San Marcos; Perú Fil: Pujos, François Roger Francis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; Argentina Fil: Ganerød, Morgan. Geological Survey of Norway; Noruega Fil: Marivaux, Laurent. Université de Montpellier; Francia

Metatherians, which comprise marsupials and their closest fossil relatives, were one of the most dominant clades of mammals during the Cretaceous and are the most diverse clade of living mammals after Placentalia. Our understanding of this group has increased greatly over the past 20 years, with the discovery of new specimens and the application of new analytical tools. Here we provide a review of the phylogenetic relationships of metatherians with respect to other mammals, discuss the taxonomic definition and diagnosis of Metatheria, outline the Cretaceous history of major metatherian clades, describe the paleobiology, biogeography, and macroevolution of Cretaceous metatherians, and provide a physical and climatic background of Cretaceous metatherian faunas. Metatherians are a clade of boreosphendian mammals that must have originated by the Late Jurassic, but the first unequivocal metatherian fossil is from the Early Cretaceous of Asia. Metatherians have the distinctive tightly interlocking occlusal molar pattern of tribosphenic mammals, but differ from Eutheria in their dental formula and tooth replacement pattern, which may be related to the metatherian reproductive process which includes an extended period of lactation followed by birth of extremely altricial young. Metatherians were widespread over Laurasia during the Cretaceous, with members present in Asia, Europe, and North America by the early Late Cretaceous. In particular, they were taxonomically and morphologically diverse and relatively abundant in the Late Cretaceous of western North America, where they have been used to examine patterns of biogeography, macroevolution, diversification, and extinction through the Late Cretaceous and across the Cretaceous-Paleogene (K-Pg) boundary. Metatherian diversification patterns suggest that they were not strongly affected by a Cretaceous Terrestrial Revolution, but they clearly underwent a severe extinction across the K-Pg boundary.

Mammals and birds are the only representatives of the Animal Kingdom able to maintain a fairly constant body temperature regardless of the temperature of the external environment. Such animals are not mere creatures of the environment but are able to carry on their usual activities under a wide range of external temperatures. From the standpoint of evolution, the warm-blooded condition is the more recently developed.Although this state of homeothermy has been realised for a considerable time, no serious attempt has been made, as yet, to carry out a systematic study of species differences In thermoregulation with regard to either degree of constancy or methods adopted. This thesis is offered in an attempt to remedy this deficiency to some extent.In no other class of animals is the process of evolution more completely Illustrated than in the mammals. Grouped within the Class Mammalia and illustrating its comprehensive nature, we have the primitive egg-laying forms of the platypus and the echidna; the marsupials of Australia and America; the rodents, the hoofed grass-eaters, the carnivores, insectivores and bats, and lastly monkeys, apes and man.The geographical distribution of mammals is most interesting; it being quite apparent that the various parts of the earth's surface, at the present day, differ greatly in respect to their animal inhabitants. The most striking feature of the Australian region (which includes Australia, Tasmania and the Austro-Malayan Islands) is the almost total absence of higher mammals or Eutheria amongst its native fauna - the mammals belonging mainly to the marsupials (Metatheria) and monotremes (Protheria). The last named Order is entirely confined to this region, whilst marsupials occur elsewhere only in America. The abundance of marsupials is quite remaikable, all the leading groups of that sub-class, with the exception o f the Dldelphyidae or American opossums being strictly endemic. .......

Femora referable to metatherians and eutherians recovered from the Bissekty Formation, Dzharakuduk, Kyzylkum Desert, Uzbekistan (90 Mya), are described. Fourteen isolated specimens were sorted based on size and morphology into groups that likely correspond to the species level or higher. Groups were then tentatively assigned to taxa known from teeth, petrosals, and/or other postcrania at these localities. One distal femur of a small arboreal metatherian, and several eutherian distal femora that probably represent zhelestids and/or zalambdalestids were identified. With the exception of one proximal femur that is similar in some aspects to the zalambdalestid Barunlestes, and a previously described multituberculate specimen, all other proximal femora from the Bissekty Formation exhibit a metatherian-like morphology. The dental record currently suggests the presence of twelve eutherian species and only one metatherian at Dzharakuduk, whereas the humeral and crurotarsal evidence supports the presence of at lea...

The Trinity therians have long been the focus of attempts to reconstruct the evolutionary history of higher mammals, es− pecially in the context of the development of tribospheny. In this paper, we update the taxonomy of the tribosphenidan taxa known from the Trinity Group and establish with more confidence the premolar/molar count in each. Many isolated specimens can be referred to a specific tooth locus. Additional diversity is revealed within the Deltatheroida, with the de− scription of an additional species of Oklatheridium; Pappotherium is here considered a likely metatherian based on the in− ferred presence of four molars, while Holoclemensia is a basal eutherian (the opposite of some traditional interpretations). The remainder of the genera, Kermackia and Slaughteria, cannot be allied with either of the living groups of tribo− sphenidan mammals using the available data. We identify strong morphological diversity within this assemblage of stem taxa, including modifications to the traditional tribosphenic occlusal pattern in Kermackia. Mammalian evolution at the base of the tribosphenidan radiation was complex, and this underscores the need for caution when interpreting the mor− phology and relationships of taxa known by incomplete material.

zoic and Paleogene †Multituberculata), and Theria. The last subclass included the infraclasses †Pantothe� ria (Mesozoic orders of †Pantotheria and †Symmetr� odonta), Metatheria (marsupials), and Eutheria (pla� centals). Mesozoic triconodonts (order †Tricon� odonta) were referred to as Mammalia incertae subclassis. The classification of Mesozoic mammals proposed by Simpson was based exclusively on struc� tural features of their molars. In the midtwentieth cen� tury, Late Triassic–Early Jurassic mammal faunas were discovered, which included taxa with tricon� odont and symmetrodont types of molars combined with primitive morphology of the lower jaw, which, along with the dentary, included postdentary and paradentary bones, suggesting the presence in these taxa of a rudimentary reptilian jaw joint between the quadrate and articular. In the modern classification of Mesozoic mammals based on cladistic analysis of 275 morphological characters [2], the class Mamma� lia is divided into three subclasses: Australosphenida (orders †Ausktribosphenida and Monotremata), †Allotheria (orders †Haramiyida and †Multitubercu� lata), and Boreosphenida (infraclasses Metatheria and Eutheria). Many Mesozoic groups, including tricon� odonts and symmetrodonts, with the double jaw joint (†Morganucodonta and †Kuehneotheriidae, respec� tively), have not been assigned to any of these sub� classes. This system postulates homoplasy in the development of certain characters, such as appearance

Sulestes karakshi Nessov, 1985b (= Deltatheroides kizylkumensis Nessov, 1993 = Marsasia aenigma Nessov, 1997) from the Late Cretaceous (Turonian) Bissekty local fauna, Kyzylkum Desert, Uzbekistan, is revised based on additional material from the type locality. It is characterized by an absence of palatal vacuities, double-rooted P1, an asymmetrical M3 with reduced metastylar lobe, an unreduced M4 and m4, obliquely oriented p1, anterior wall of the upper canine alveolus formed by premaxilla, and Meckelian groove on the dentary. PAUP analyses using a data matrix modified from Rougier et al. (1998, 2004) places Sulestes within Deltatheridiidae in an unresolved trichotomy with the Mongolian Campanian Deltatheridium and Deltatheroides. Oklatheridium from the Early Cretaceous of North America is sister taxon to these Late Cretaceous Asiatic deltatheridiidans. Deltatheridiidae is the sister group to other Metatheria including the crown clade Marsupialia. A Deltatheroides-like taxon from the Maastrichtian at Guri...

Deltatheroida is a small, mostly Asian, clade of Cretaceous mammals with a complicated systematic history and a poor North American fossil record. The recently described latest Cretaceous Nanocuris improvida is a relatively large, morphologically distinctive taxon with carnivorous adaptations; it was tentatively referred to its own family Nanocuridae within Eutheria. Here, we describe a new specimen from the uppermost Cretaceous Lance Formation of Wyoming that is referable to Nanocuris improvida, the second occurrence for the taxon. New morphological information from the specimen allows us to refer five additional Lancian specimens, some of which have been previously described as deltatheroidans, to Nanocuris improvida and ?Nanocuris sp. Results from a phylogenetic analysis remove Nanocuris from Eutheria and reveal strong support for its deltatheroidan affinities and for its nested position within Metatheria. As a result, we subsume Nanocuridae within the deltatheroidan family Deltatheridiidae. W...

Based on recent hypotheses on mammalian phylogeny, the opening of the vomeronasal organ (VNO) was found to be located in the naso-palatine duct (DNP) or caudal to its anterior margin in the therian morphotype, whereas it is located rostral of the DNP in the glirean morphotype. These conclusions are derived in a successive reconstruction of the morphotypes of all higher eutherian and metatherian taxa determined from own and literature data. The three data sets examined consist of data obtained from adults, ontogenetic-sequences, and pooled pre-adult/adult data.

The Metatheria include not only marsupials but all therians more related to Marsupialia than to the Eutheria. Marsupialia is considered as a metatherian crown group including all extant marsupials, their common ancestor and all of their descendants. “Ameridelphia” is not a natural group. Australidelphia includes the Microbiotheria and all Australasian marsupials. Several authors also argue that the Polydolopimorphia are Australidelphians as well. Relationships of Sparassodonta with other Metatheria are a matter of discussion. To several authors, they are more closely related to South American and Australian groups than to basal North American and/or Asian metatherians. Our concept of Didelphimorphia includes the Peradectoidea (Peradectidae and Caroloameghiniidae) and the Didelphoidea (Didelphidae and Sparassocynidae). In several analyses, the Paucituberculata appear as more closely related to the Australidelphia than to the Didelphimorphia. The relationships of the Microbiotheria within the Australidelphia have been subject of much discussion. They have been considered either as sister-taxa of all other Australidelphia, at the base of Diprotodontia, as a sister-taxon of Dasyuromorpha, as a sister-taxon of Phalangeriformes + Diprotodontia, or even related with part of the former.

The anterior commissure (AC) and the much smaller hippocampal commissure constitute the only interhemispheric pathways at the telencephalic level in birds. Since the degeneration study from Zeier and Karten (1973), no detailed description of the topographic organization of the AC has been performed. This information is not only necessary for a better understanding of interhemispheric transfer in birds, but also for a comparative analysis of the evolution of commissural systems in the vertebrate classes. We therefore examined the fiber connections of the AC by using choleratoxin subunit B (CTB) and biotinylated dextran amine (BDA). Injections into subareas of the arcopallium and posterior amygdala (PoA) demonstrated contralateral projection fields within the anterior arcopallium (AA), intermediate arcopallium (AI), PoA, lateral, caudolateral and central nidopallium, dorsal and ventral mesopallium, and medial striatum (MSt). Interestingly, only arcopallial and amygdaloid projections were reciprocally organized, and all AC projections originated within a rather small area of the arcopallium and the PoA. The commissural neurons were not GABA‐positive, and thus possibly not of an inhibitory nature. In sum, our neuroanatomical study demonstrates that a small group of arcopallial and amygdaloid neurons constitute a wide range of contralateral projections to sensorimotor and limbic structures. Different from mammals, in birds the neurons that project via the AC constitute mostly heterotopically organized and unidirectional connections. In addition, the great majority of pallial areas do not participate by themselves in interhemispheric exchange in birds. Instead, commissural exchange rests on a rather small arcopallial and amygdaloid cluster of neurons. J. Comp. Neurol. 524:343–361, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Both metatherians and eutherians are known from the Early Cretaceous (Barremian, 125 mya; million years ago) of China, while eutherian-dominated mammalian faunas appeared in Asia at least by the earliest Late Cretaceous (Cenomanian, 95 mya). The approximately 99–93 my old (Cenomanian) Sheikhdzheili l.f. from western Uzbekistan is a small sample of only eutherians, including three zhelestids and a possible zalambdalestoid. The much better-known 90 my old (Turonian) Bissekty l.f. at Dzharakuduk iin central Uzbekistan includes 15 named and unnamed species, based on ongoing analyses. Of these, 12 are eutherians represented by at least the three groups—asioryctitheres, zalambdalestids, and zhelestids—plus an eutherian of uncertain position—Paranyctoides. Zalambdalestids and zhelestids have been argued to be related to the origin of the placental gliriforms (Euarchontoglires) and ferungulates (Laurasiatheria), respectively. Although there are four previously recognized metatherians, we believe three are referable to the deltatheroid Sulestes karakshi and the fourth, Sailestes quadrans, may belong to Paranyctoides. There is one multituberculate and one symmetrodont in the Bissekty l.f. While comparably aged (Turonian) localities in North America have somewhat similar non-therians, they have more metatherians and no eutherians. The next younger localities (early Campanian, ∼80 mya) in North America have both a zhelestid and Paranyctoides, suggesting dispersal of eutherians from Asia. At Dzharakuduk, the approximately 85 my old (late Turonian/Coniacian) Aitym l.f. is much less well known than the Bissekty l.f., but yields nearly identical taxa, with two non-therians, one metatherian, and six eutherians.

Theria includes Eutheria and its sister taxon Metatheria. Placentalia includes extant eutherians plus their most recent common ancestor. The oldest eutherian is from 125mya (million years ago). Molecular studies place this origin at about 130-185mya. Older dates cannot be refuted based on fossil evidence as earliest eutherian remains are scarce. Earliest superordinal clades (hence Placentalia) range from 64-104mya (median 84mya) based on molecules, similar to 85-90mya based on fossils. Superordinal clades Archonta, Ferungulata, Glires, and Paenungulata based on fossils are similar to molecularly based clades, except Afrotheria was not predicted by fossils. Both fossils and molecules recognize 16 of 18 extant placental orders. Fossils place the origins of orders around 65mya as do some molecular studies, but others suggest ordinal diversification as old as 100mya. Fossil evidence supports a Laurasian origin for Eutheria (and Metatheria) and Placentalia, although some molecular studies suggest a Gondwanan origin for both taxa.

Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage. Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.” DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions. The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.

Our understanding of the diversity of mammalian life histories is based almost exclusively on eutherian mammals, in which the slow–fast continuum persists even after controlling for effects of body size and phylogeny. In this paper, we use modern comparative methods to test the extent to which this eutherian-based framework can be extrapolated to metatherian mammals. First, we examine the pattern of covariation among life history traits, and second, we test for correlations between variation in life history and variation in six candidate ecological variables: type of diet, extent of intraspecific competition, risk of juvenile mortality, diurnal pattern of activity, arboreality, and rainfall pattern. Even when controlling for body size and phylogeny, we observe a slow–fast continuum in metatherian mammals. Some parameters involved are different from those identified by studies of eutherians, but the underlying relationships among longevity, fecundity, and age at maturity persist. We also show that overall variation in a key life history variable, reproductive output (measured by annual reproductive rate and litter size), is significantly related to variation in type of diet, with a foliage-rich diet being associated with low fecundity. This is interesting because, although ecological correlations have been found within some eutherian subgroups, modern comparative approaches have failed to reveal robust ecological correlates of overall life history diversity in eutherians.

Extant eutherian mammals and their most recent common ancestor constitute the crown group Placentalia. This taxon, plus all extinct taxa that share a more recent common ancestor with placentals than they do with Metatheria (including marsupials), constitute Eutheria1. The oldest well documented eutherian-dominated fauna in the world is Dzharakuduk, Uzbekistan2. Among eutherians that it yields is Kulbeckia, an 85–90-Myr-old member of Zalambdalestidae (a family of Late Cretaceous Asian eutherians)3. This extends Zalambdalestidae back by some 10 million years from sites in the Gobi Desert, Mongolia4. A phylogenetic analysis of well described Late Cretaceous eutherians strongly supports Zalambdalestidae, less strongly supports ‘Zhelestidae’ (a Late Cretaceous clade related to Tertiary ungulates), but does not support Asioryctitheria (a group of Late Cretaceous Asian eutherians). A second analysis incorporating placentals from clades that include rodents (Tribosphenomys), lagomorphs (Mimotona) and archaic ungulates (Protungulatum and Oxyprimus) strongly supports Zalambdalestidae in a clade with Glires (rabbits, rodents and extinct relatives) and less strongly ‘Zhelestidae’ within a clade that includes archaic ungulates (‘condylarths’). This argues that some Late Cretaceous eutherians belong within the crown group Placentalia. The ages of these taxa are in line with molecularly based estimates of 64–104 Myr ago (median 84 Myr ago) for the superordinal diversification of some placentals5, but provide no support for a Late Cretaceous diversification of extant placental orders.

Paleostratigraphic estimates of divergence time for nine independent cladogenic events within Mammalia, ranging from 14 to 130 million years, were regressed against Tamura–Nei-corrected 12S rRNA transversions. Relative rate-adjusted distances were also regressed against paleostratigraphic divergence times. The resulting equations were used to estimate interordinal divergence times within Eutheria and Metatheria for a data set that includes representatives of all orders in each infraclass. Without the adjustment for rate variation, divergence times range from 34 to 156 million years for placental orders, versus 32 to 86 million years for marsupial orders. With rate adjustments, the range of divergence estimates decreases to 53 to 133 million years for placentals versus 40 to 79 million years for marsupials. The effect of rate adjustments is most noticeable for carnivores and perissodactyls, where rates are slow, and proboscideans, where rates are fast. In agreement with studies based on nuclear genes, both unadjusted and rate-adjusted estimates of sequence divergence indicate that the majority of placental orders originated before the terminal Cretaceous extinction. Exceptions include the perissodactyl–carnivore split and cladogenesis among paenungulate orders. Most marsupial orders, in turn, may have originated in the early Tertiary although didelphimorphs, at least, appear to have split from other lineages in the late Cretaceous. Marsupial divergence times based on 12S rRNA data are in good agreement with estimates based on single-copy DNA hybridization and disagree with the suggestion of Hershkovitz (1992) that Dromiciops separated from other marsupials in the Jurassic.

A marsupial mammal from Mongolia representing a previously unreported group, Asiadelphia, offers unequivocal evidence that metatherians were represented on the continent of Asia during the Late Cretaceous (Campanian), even in the northern arid interior. Asiatherium is known by skull, left and right mandibles, and most of its postcranial skeleton. Comparisons of the dentition, ear region, and aspects of the postcranium not only allow its diagnosis as an Asian group of metatherians but also permit a diagnosis of the Metatheria based on the unequivocally apomorphous condition of the dental formula and probable replacement pattern of the protometatherian, in contrast to the more primitive therian dental formula, represented by the few known Aegialodontidae and the first dental eutherians. It appears an inescapable conclusion that the first metatherians had a more derived dental formula (and probably replacement pattern) than the earliest dental eutherians, regardless of what specific derivation from a therian ancestry is contemplated. Such a diagnosis also supports the metatherian status of other Cretaceous Asian and American taxa such as the Deltatheroida and Kokopellia. The hypothesis that Metatheria originated in North America is largely dependent on the preponderance of Cretaceous forms from North America and negative evidence from Asia (i.e., on the lack of lower latitude Early Cretaceous tropical faunas). Conversely, the relative Cretaceous paucity of placentals from North America and their greater abundance in Asia suggests the earliest flowering of the Eutheria in an unspecified region of the Old World. The concept of a holophyletic Theria (= Tribosphenida) employed here, based on the biologically significant apomorphy of the molar form-function of the first therian, entails only the tribosphenic mammals (infraclasses Tribotheria, Metatheria, and Eutheria) but not the sundry quasi-triangular-toothed mammals and their derivatives in the Mesozoic (Eupantotheria, Peramura, Monotremata, etc.). It is suggested that the Asiadelphia and Deltatheroida, the protospecies of both, with the apomorphic therian dental formula, are ancient lineages of Metatheria independent from their North American relatives probably since the early part of the Late Cretaceous.

The Y chromosome is known to induce testis differentiation in Eutheria and Metatheria. Recently, in Eutheria, a gene ( Sry ), which is the major candidate to be the testis-determining gene, has been identified. We present evidence indicating that DNA sequences homologous to Sry are male specific in two marsupial species. This finding leads us to conclude that Sry also is the testis-determining gene in Metatheria.

A cladistic analysis of the major groups of early metatherian mammals shows that the Deltatheroida and South American dog-like Borhyaenoidea are closely related and are placed in a new supercohort Deltatheralia. while all other metatherians are placed in the supercohort Marsupialia. A reassessment of Early Cretaceous mammals with tribosphenic molars shows that metatherians and eutherians apparently evolved independently from a peramuran-like ancestor, and an entoconid. distinctly basined talonid and large protocone were acquired separately in each group. Consequently. the Tribosphenida (sensu McKenna) is apparently paraphyletic. Aegialodontia are regarded as structurally ancestral to Metatheria but not Eutheria. The known fossil record supports the view that differentation of the stocks which gave rise to metatherians and eutherians apparently occurred in Late Jurassic or Early Cretaceous time.

Polyphyletic or monophyletic origins of mammals have been subjects of considerable controversy for a century. Late Triassic—Early Jurassic mammals are more diverse than previously thought. The stumbling block in establishing mono‐ or polyphyletic origins of mammals is the uncertain position of the Haramiyidae (the oldest Multituberculata, and possibly the oldest mammals), known only from isolated teeth. Triconodonta and Theria probably shared an unknown common ancestor, while Monotremata possibly branched from early Eupantotheria at the end of the Early Jurassic, before the Dryolestoidea did, from the forms in which the cochlea started to coil and small cerebellar hemispheres developed. Aegialodontia gave rise to Metatheria but not to Eutheria. Deltatheroida belongs to Metatheria. A group of Cretaceous therian mammals with tribosphenic molars informally classified as “Tribotheres”; cannot be assigned either to Metatheria or Eutheria.

The major lineages of mammals (Eutheria, Metatheria, and Monotremata) diverged more than 100 million years ago and have undergone independent changes in the neocortex. We found that adult South American gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii) possess a significantly lower number of cerebral cortical neurons compared with the mouse (Mus musculus). To determine whether the difference is reflected in the development of the cortical germinal zones, the location of progenitor cell divisions was examined in opossum, tammar wallaby, and rat. The basic pattern of the cell divisions was conserved, but the emergence of a distinctive band of dividing cells in the subventricular zone (SVZ) occurred relatively later in the opossum (postnatal day [P14]) and the tammar wallaby (P40) than in rodents. The planes of cell divisions in the ventricular zone (VZ) were similar in all species, with comparable mRNA expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in opossum (P12 and P20) and mouse (embryonic day 15 and P0). In conclusion, the marsupial neurodevelopmental program utilizes an organized SVZ, as indicated by the presence of intermediate (or basal) progenitor cell divisions and gene expression patterns, suggesting that the SVZ emerged prior to the Eutherian-Metatherian split.

The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5′ distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORESINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution. © 2007 Santangelo et al. Fil:Santangelo, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rubinstein, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

The class mammalia is composed of approximately 4800 extant species. This class is divided into three subclasses, the prototheria (monotremes), metatheria (marsupials), and eutheria. Surprisingly, there is relatively little knowledge about germ layer and axis formation in mammalian species. Most knowledge about these embryonic processes has been obtained from one species, the mouse, Mus musculus. Here we discuss major variations in germ layer and axis formation among mammals. We suggest that more studies of embryonic development in diverse mammalian species are required for an understanding of germ layer and axis formation to provide insights into human biology and disease.

Derived features of a new boreosphenidan mammal from the Lower Cretaceous Yixian Formation of China suggest that it has a closer relationship to metatherians (including extant marsupials) than to eutherians (including extant placentals). This fossil dates to 125 million years ago and extends the record of marsupial relatives with skeletal remains by 50 million years. It also has many foot structures known only from climbing and tree-living extant mammals, suggesting that early crown therians exploited diverse niches. New data from this fossil support the view that Asia was likely the center for the diversification of the earliest metatherians and eutherians during the Early Cretaceous.

Ranging in size from a shrew to the blue whale, mammals are found in the air, on and under the ground, and in the oceans, rivers, and lakes of the world. A total of 151 families are divided into 1226 genera and represent 5416 species. The mammalian middle ear is a key diagnostic character and has evolved from three small bones found in the jaw of primitive vertebrates. Mammals regulate their internal temperature (endothermy). Diagnostic features include: sweat glands, hair, a complex lung structure, and mammary glands: the production of milk to feed and nurture young is a shared feature of all mammals.

The earliest mammals were all small, nocturnal insectivorous creatures, and it appears that the basic mode of mammalian reproduction remained unchanged from the time of the origin of mammals in the late Triassic (about 200 million years ago) until the dispersal of the Gondwanan continents in the middle Cretaceous (95-110 million years ago). This meant that there was no land faunal communication between Africa and the Antarctic-Australia-India complex except via South America after the earliest Cretaceous (Lillegraven et al., 1979). The major adaptive radiations of the advanced therian mammals occurred during the middle Cretaceous, so that by the late Cretaceous (75-80 million years ago) the Eutheria and Metatheria were already distinct and largely geographically isolated (Archer, 1984; Tyndale-Biscoe and Renfree, 1987). Whilst marsupials are true mammals in every sense, and in endocrine terms have the same range of hormones, these hormones are sometimes used in different ways (Amoroso et al., 1980) - for example, prolactin inhibits the marsupial corpus luteum, whereas it is luteotrophic in some eutherian mammals. This communality of hormones but diversity of actions between the mammalian groups clearly demonstrates the validity of Medawar’s (1953) aphorism that it is not hormones that have evolved, but rather the uses to which they are put.