Limpets break Dollo's Law

A new molecular phylogeny of the limpet molluscs (Calyptraeidae) reveals that coiled shells have independently re-evolved at least once in this family, which is a violation of Dollo's Law that complex ancestral states, once lost, are never reacquired. Reacquisition of the coiled ancestral state is remarkable in that uncoiled shells have been the most recent ancestral state for 20 million-100 million years. Adult coiling might have re-evolved by the mechanism of prolonging the period during which genes for coiling are expressed in larvae. This and other developmental mechanisms could provide general routes for maintaining the potential to produce traits lost in distant ancestors.     

Upper Cretaceous rudist biostratigraphy of the Bey Da?lar? Carbonate Platform, Western Taurides, SW Turkey

The Upper Cretaceous (Middle Cenomanian-Coniacian) successions of the Bey Da?lar? Carbonate Platform (Western Taurides, SW Turkey) are represented by rudist-bearing shallow-water limestones. Four rudist lithosomes are distinguished for the first time from the Eastern, Northern and Southern Areas of the Bey Da?lar? Autochthon. The oldest rudist assemblages dominated by caprinids are observed in the Eastern (Katran Da?) Area (caprinid lithosomes) and suggest a Middle-Late Cenomanian age. The uppermost part of the platform carbonates in the Northern Area is characterized by an association of hippuritid and radiolitid rudist bivalves dominated by Vaccinites praegiganteus (Toucas) (hippuritid lithosomes). The rudist fauna indicates the Late Turonian age, which is confirmed by the previously obtained ⁸⁷Sr/⁸⁶Sr values of well-preserved low-Mg calcite of Vaccinites praegiganteus (Toucas) shells. The rudist associations of the Southern (Susuzda?) Area are represented by two rudist formations. The lower lithosomes are mainly made up of hippuritids and radiolitids (hippuritid-radiolitid lithosomes). The stratigraphical distributions of the species of the assemblage indicate a Santonian-Early Campanian age. The rudist associations of the upper lithosomes are dominated by species of Joufia and Gorjanovicia (Joufia-Gorjanovicia lithosomes), which suggest a Late Campanian-Maastrichtian age. Identification of the rudist lithosomes yields information on the palaeobiogeographic distribution of the rudist species in the eastern Mediterranean region and also on the biostratigraphic frame of the Upper Cretaceous successions of the Bey Da?lar? Carbonate Platform.     

Strontium isotope stratigraphy of Cretaceous hippuritid rudist bivalves: rates of morphological change and heterochronic evolution

Strontium isotope stratigraphy of 17 localities of rudist formations in the region of the former Mediterranean Tethys has provided a reliable and precise stratigraphical frame for the evaluation of morphological change in hippuritid rudist bivalves during the Coniacian–Campanian. The phyletic lineage Vaccinites cornuvaccinum (Bronn)–Vaccinites chaperi (Douvillé) evolved from the Early Coniacian until the Early Campanian and is characterized by phyletic size increase and allometric growth, as shown by morphometrical measurements of 102 shells. These chronospecies intergrade in the Late Coniacian so that V. cornuvaccinum is considered to be a reliable marker species for the Coniacian. The taxonomy of Vaccinites alpinus (Douvillé) is discussed and the species is recognized as a senior synonym of Vaccinites ultimus (Milovanovi ). It appears first in the Late Santonian and the last appearance is probably in Late Campanian. Both lineages are characterized by phyletic size increase and peramorphic evolution involving hypermorphosis. A doubling of the length of the mantle margin occurred within 5 m.y. in both lineages. The results demonstrate that the combination of morphometric analyses and stratigraphical precision provides an important tool for the delineation of tempo and mode of evolution in rudist bivalves. Strontium isotope stratigraphy resulted in a considerable revision of the ranges of the species investigated. As the stratigraphy of many Tethyan carbonate platforms relies on the distribution of rudist bivalves, and the species investigated are abundant in many rudist formations, the history of many Late Cretaceous carbonate platforms must be re-evaluated.     

Shell disintegration and taphonomic loss in rudist biostromes

Radiolitid biostromes in the Upper Cretaceous of Austria and Italy record a marked taphonomic loss controlled mainly by the composition of the biocoenosis, by the density of rudist colonization, by the style of radiolitid shell disintegration and by early diagenetic processes. Radiolitid shells consisted of a calcitic ostracum and an originally aragonitic hypostracum. The attached valve of most radiolitids was built of (1) an outermost ostracal layer of delicate calcite lamellae, (2) a thick layer of ‘boxwork ostracum’ built of radial funnel plates and cell walls, (3) a thin, inner ‘ostracal layer 3’ of thick-walled boxwork, and (4) the hypostracum that formed the innermost shell layer. The attached valve disintegrated by spalling of radial funnel plates of layer 2, and by selective removal of the boxwork ostracum. In the free valve, the ostracum consisted of two layers: (a) an inner, lid-shaped layer of dense calcite, and (b) an outer layer composed of calcite lamellae. The free valve disintegrated by spalling into ostracal and hypostracal portions, by spalling of the ostracum into layers a and b, and by disintegration of layer b into packages of calcite lamellae and individual lamellae. The specific style of disintegration of the radiolitids was aided or induced by discontinuities in shell structure. Lamellar fragments from the ostracum of the upper valve and from the radial funnel plates of the lower valve locally are abundant in free-valve-funnel-plate floatstones that comprise the matrix of or occur in lenses within radiolitid biostromes. In biostromes with an open parautochthonous fabric, selective removal of the boxwork ostracum of the attached valve occurred by mechanical spalling and, most probably, by early diagenetic dissolution. Complete removal of the boxwork ostracum yielded thin, relict shells composed of the ‘ostracal layer 3’ and the hypostracum. During early diagenesis, the hypostracum was replaced by blocky calcite spar, or was dissolved and became filled by internal sediments. The combination of both selective removal of boxwork ostracum and early diagenetic dissolution of aragonite locally resulted in the formation of ghost biostromes that entirely or largely consist of faint relics of radiolitids. The syndepositional formation of radiolitid shell relics and the presence of radiolitid ghost biostromes produced by bios-tratinomic and early diagenetic processes show that rudist biostromes can undergo marked taphonomic loss during fossilization. The presence of ghost biostromes with a burrowed, open parautochthonous rudist fabric indicates that the final preservation of a rudist biostrome was directly influenced by the characteristics of the biocoenosis, including unpreserved burrowing taxa. Rudist biostromes may be of markedly different taphonomy as a result of the taxonomic composition of the entire assemblage and the density of colonization by the rudists.     

Dollo's law and the re-evolution of shell coiling

Gastropods have lost the quintessential snail feature, the coiled shell, numerous times in evolution. In many cases these animals have developed a limpet morphology with a cap-shaped shell and a large foot. Limpets thrive in marginal habitats such as hydrothermal vents, the high-energy rocky intertidal areas and fresh water, but they are considered to be evolutionary dead-ends, unable to re-evolve a coiled shell and therefore unable to give rise to the diversity seen among coiled snails. The re-evolution of a coiled shell, or any complex character, is considered unlikely or impossible (Dollo's law) because the loss of the character is followed by the loss of the genetic architecture and developmental mechanisms that underlie that character. Here, we quantify the level of coiling in calyptraeids, a family of mostly uncoiled limpets, and show that coiled shells have re-evolved at least once within this family. These results are the first demonstration, to our knowledge, of the re-evolution of coiling in a gastropod, and show that the developmental features underlying coiling have not been lost during 20-100 Myr of uncoiled evolutionary history. This is the first example of the re-evolution of a complex character via a change in developmental timing (heterochrony) rather than a change in location of gene expression (heterotopy).     

Palaeoecological significance of Barremian ammonite assemblages and facies variations from Southwest Mexico

Analyses of ammonite shell forms of two Barremian stratigraphic sections from Southwest Mexico consist of two well-defined morphotypes: (1) Small uncoiled, mostly leptoceratoid ancyloconic shells of the families Ancyloceratidae and Hamulinidae, and (2) middle-sized involute to moderately evolute oxycone to discocone shells of the family Pulchelliidae. Index taxa allow the recognition of standard ammonite biozones for the Barremian, which permit the relative dating of different processes that occurred through the water column in the environment of deposition. The vertical distribution of ammonite morphotypes and facies suggests changes of the palaeoceanographic and sedimentological conditions that prevailed in the area during Barremian time. Petrologic data, analyses of the organic carbon and carbonate contents of the rocks support the idea that oxygen-deficient bottom waters existed within a shallow marine, tectonically active area with little carbonate deposition during the early early Barremian (upper part of the Taveraidiscus hugii Zone through the base of the Nicklesia pulchella Zone). These conditions in the basin caused a proliferation of middle-water depth ammonites of Morphotype 1 but prevented the abundance of nektobenthic forms of Morphotype 2. Oxic conditions on a more calcareous and open normal marine environment seem to have been reestablished progressively during a transgressive episode from late early-early late Barremian (upper part of the Nicklesia pulchella Zone through the Gerhardtia sartousiana Zone). This environmental setting supported more facies dependent nektobenthic ammonites of Morphotype 2 to flourish within the basin.     

Cenomanian carbonate facies and rudists along shallow intraplatform basin margin-the island of Cres (Adriatic Sea, Croatia)

Summary The island of Cres is located in the northern part of the Adriatic Sea. The island is built up of predominantly Cretaceous carbonates deposited in north-western part of extensive and long-lasting Adriatic Carbonate Platform. Owing to the influence of synsedimentary tectonics supported by eustatic changes during the latest Albian/Early Cenomanian, different sedimentary environments were established: from shallow intraplatform basin and related slope, across basin margin to protected shallow-platform. During the Early to Middle Cenomanian rudist communities (ichthyosarcolitid/caprinid/radiolitid)flourished along a relatively high-energy intraplatform basin margin. Fair amounts of coarse-grained bioclasts, derived almost exclusively from broken rudist shells, were deposited over a marginal depocenter. Contemporaneously, pithonellid wackestone-packstones containing microbioclasts and planktonic foraminifera were deposited basinward while marginal bioclastic sediments and limestone blocks of the basin margin origin were sporadically deposited within the basin. The opening of the Cres intraplatform basin was aborted and the basin was finally filled up during the Late Cenomanian. Since the Cres intraplatform basin was established at the beginning of the Cenomanian it probably represented the initiation phase in the north-western extension of the later Adriatic Trough development.     

DOES DIVING LIMIT BRAIN SIZE IN CETACEANS?

We test the longstanding hypothesis, known as the dive constraint hypothesis, that the oxygenation demands of diving pose a constraint on aquatic mammal brain size.Using a sample of 23 cetacean species we examine the relationship among six different measures of relative brain size, body size, and maximum diving duration. Unlike previous tests we include body size as a covariate and perform independent contrast analyses to control for phylogeny. We show that diving does not limit brain size in cetaceans and therefore provide no support for the dive constraint hypothesis. Instead, body size is the main predictor of maximum diving duration in cetaceans. Furthermore, our findings show that it is important to conduct robust tests of evolutionary hypotheses by employing a variety of measures of the dependent variable, in this case, relative brain size.     

The evolution of centipede venom claws – Open questions and possible answers

The maxilliped venom claw is an intriguing structure in centipedes. We address open questions concerning this structure. The maxillipeds of fossil centipedes from the Carboniferous (about 300 million years old) have been described, but not been depicted previously. Re-investigation demonstrates that they resemble their modern counterparts. A Jurassic geophilomorph centipede (about 150 million years old) was originally described as possessing a rather leg-like maxilliped. Our re-investigation shows that the maxilliped is, in fact, highly specialized as in modern Geophilomorpha. A scenario for the evolution of the centipede maxilliped is presented. It supports one of the two supposed hypotheses of centipede phylogeny, the Pleurostigmophora hypothesis. Although this hypothesis appears now well established, many aspects of character evolution resulting from this phylogeny remain to be told in detail. One such aspect is the special joint of the maxilliped in some species of Cryptops. Cryptops is an in-group of Scolopendromorpha, but its maxilliped joint can resemble that of Lithobiomorpha or even possess a mixture of characters between the both. Detailed investigation of fossils, larger sample sizes of extant species, and developmental data will be necessary to allow further improvements of the reconstruction of the evolutionary history of centipedes.     

Ancestral transfer of symbionts between cockroaches and termites: an alternative hypothesis.

Closely related cellulolytic protozoa reside in the hindguts of extant woodroaches (Cryptocercidae) and termites (Isoptera). The evolutionary origin of these symbiotic relationships in the two lineages is uncertain. Transfer of protozoa between ancestors of modern Cryptocercus and termites remains a valid alternative theory to the established hypothesis of symbiont inheritance from a common ancestor. Nalepa's (Proc. R. Soc. Lond. B 246, 185 (1991] concerns regarding the protozoan transfer hypothesis focus on the biology of modern species, and neglect to consider the evolutionary framework of an ancestral dynamic postulated to occur among Palaeozoic insects. Legitimacy of the symbiont transfer theory removes the constraint of interpreting presence of cellulolytic protozoa as a synapomorphy between Cryptocercidae and Isoptera, with potential impact on objective resolution of dictyopteran phylogeny.     

An Ancient Repeat Sequence in the ATP Synthase β-Subunit Gene of Forcipulate Sea Stars

A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase β-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

COMPUTER-SIMULATED SHELL SIZE AND SHAPE VARIATION IN THE CARIBBEAN LAND SNAIL GENUS CERION: A TEST OF GEOMETRICAL CONSTRAINTS

A computer graphical model of gastropod shell form is used to test a hypothesis of geometric constraint proposed to explain the disjunct distribution of shell forms observed in Cerion, a species-rich and geometrically varied genus of terrestrial gastropods. The mapping of computer-simulated forms into a morphospace of Cerion shells produces a continuum of sizes and shapes. Therefore, the absence of particular shell forms is not explained by geometric constraints. Two proposed modes of shell morphogenesis at extreme ranges in size (“dwarfs” and “giants”) previously were thought to be exclusive routes to the construction of high-spired (“smokestack”) forms. The present study shows that there are, in fact, multiple routes of transformation. In addition, these routes are geometrically reversible and interconnect the members of the shell-form continuum. Thus, the possible pathways followed during the course of evolution within this genus cannot be determined until an adequate phylogenetic hypothesis has been proposed.     

Evolution in isolation: the Gyraulus species flock from Miocene Lake Steinheim revisited

The Miocene Steinheim Basin in SW Germany is an ancient (long-lived) palaeo-lake that has existed over some hundreds of thousands of years. It is an iconic fossil site, because the historically oldest phylogenetic tree of extinct organisms was based on specimens described from this locality. Today the basin contains 30–40 m thickness of lake sediments with planorbid snails of the genus Gyraulus occurring in rock-forming quantities. The shells are morphologically highly disparate with forms ranging from the tiny, planispiral founder species Gyraulus kleini, to fragile corkscrew-like uncoiled forms and to large trochiform morphs with thick shells. In total, this presumably monophyletic species flock contains 17 species distributed in time and space, all of which are endemic, except for the founder species. Up to nine of them occur in a single sedimentary level and are inferred to have lived together. Such an extreme rate of endemism makes fossil Lake Steinheim special among extant and fossil lakes. This review article summarises and discusses the species concept(s), indications for endemism, speciation processes, the phylogenetic concept(s) and factors controlling evolution. It also provides directions for future research.     

Phylogenies of Flying Squirrels (Pteromyinae)

The phylogeny of flying squirrels was assessed, based on analyses of 80 morphological characters. Three published hypotheses were tested with constraint trees and compared with trees based on heuristic searches, all using PAUP*. Analyses were conducted on unordered data, on ordered data (Wagner), and on ordered data using Dollo parsimony. Compared with trees based on heuristic searches, the McKenna (1962) constraint trees were consistently the longest, requiring 8–11 more steps. The Mein (1970) constraint trees were shorter, requiring five to seven steps more than the unconstrained trees, and the Thorington and Darrow (2000) constraint trees were shorter yet, zero to one step longer than the corresponding unconstrained tree. In each of the constraint trees, some of the constrained nodes had poor character support. The heuristic trees provided best character support for three groups, but they did not resolve the basal trichotomy between a Glaucomys group of six genera, a Petaurista group of four genera, and a Trogopterus group of four genera. The inclusion of the small northern Eurasian flying squirrel, Pteromys, in the Petaurista group of giant South Asian flying squirrels is an unexpected hypothesis. Another novel hypothesis is the inclusion of the genus Aeromys, large animals from the Sunda Shelf, with the Trogopterus group of smaller "complex-toothed flying squirrels" from mainland Malaysia and southeast Asia. We explore the implications of this study for future analysis of molecular data and for past and future interpretations of the fossil record.     

Evolution and biomineralization of pteropod shells

Shelled pteropods, known as sea butterflies, are a group of small gastropods that spend their entire lives swimming and drifting in the open ocean. They build thin shells of aragonite, a metastable polymorph of calcium carbonate. Pteropod shells have been shown to experience dissolution and reduced thickness with a decrease in pH and therefore represent valuable bioindicators to monitor the impacts of ocean acidification. Over the past decades, several studies have highlighted the striking diversity of shell microstructures in pteropods, with exceptional mechanical properties, but their evolution and future in acidified waters remains uncertain. Here, we revisit the body-of-work on pteropod biomineralization, focusing on shell microstructures and their evolution. The evolutionary history of pteropods was recently resolved, and thus it is timely to examine their shell microstructures in such context. We analyse new images of shells from fossils and recent species providing a comprehensive overview of their structural diversity. Pteropod shells are made of the crossed lamellar and prismatic microstructures common in molluscs, but also of curved nanofibers which are proposed to form a helical three-dimensional structure. Our analyses suggest that the curved fibres emerged before the split between coiled and uncoiled pteropods and that they form incomplete to multiple helical turns. The curved fibres are seen as an important trait in the adaptation to a planktonic lifestyle, giving maximum strength and flexibility to the pteropod thin and lightweight shells. Finally, we also elucidate on the candidate biomineralization genes underpinning the shell diversity in these important indicators of ocean health.     

Polygyrid land snail phylogeny: external sperm exchange, early North American biogeography, iterative shell evolution

A fresh assessment of reproductive behaviour and anatomy, combined with new allozyme data, results in a phylogenetic hypothesis for the 23 polygyrid genera that differs substantially from previous attempts. Analyses based on this phylogeny suggest that (a) evolutionary transition from internal to external sperm exchange occurred only once in polygyrids, with unusual functional intermediates still extant; (b) polygyrid biogeographic history paralleled that of plethodontid salamanders, with hypothesized vicariance events at about 145, 120, 65 and 40 MA, and, dispersal events at about 55 and 40 MA; and (c) iterative shell evolution occurred despite a phylogenetic constraint on ontogenetic whorl-expansion rate.     

Mode of life of the Silurian uncoiled gastropod Semitubina sakoi n. sp. from Japan

Semitubina sakoi n. sp. from the late Silurian of Japan represents the second species of this genus and also the first record of a Silurian gastropod in Japan. The gastropod shells occur in a thin mudstone bed and were found to be encrusted exclusively by corallites of ? Favosites sp. These corallites reveal that encrustation proceeded as the gastropod shells grew. The ecological relationship between the two organisms is considered to be symbiotic. This mode of life allowed the coral to live on a muddy substrate because clear sea water passed over the colony as the gastropod moved along. The gastropod benefited from this relationship by being protected from shell-boring or shell-crushing predators by the encrusting corallite. In Semitubina sakoi the body whorl is separated from the penultimate one by a considerable gap in a later growth stage and S. sakoi has been cited as one of the uncoiled gastropods. The uncoiling of this gastropod results primarily from rapidly increasing whorl translation rate in the latest growth stage. Taking the symbiotic relationship with ? Favosites sp. into consideration, a deposit feeding or benthic scavenging mode of life is suggested for this gastropod.     

Maastrichtian facies succession and sea-level history of the Hossein-Abad,Neyriz area,Zagros Basin

The Maastrichtian shallow-water carbonate platform (Tarbur Formation) is described from outcrop in southwest Iran. It is characterised by eight microfacies types, which are dominated by larger foraminifera, rudist debris and dasycladacean algae. They are grouped into four distinct depositional settings: tidal flat, lagoon, barrier and open marine. The depositional settings include stromatolitic boundstone of tidal flat, peloidal dasycladacean miliolids wackestone and peloid bioclastic imperforate foraminifera wackestone of restricted lagoon, Omphalocyclus miliolids bioclast packstone–grainstone and miliolids intraclast bioclast packstone–grainstone of open lagoon, rudist bioclast grainstone of inner-platform shoals and rudist bioclast floatstone–rudstone and bioclastic wackestone of open-marine environments.

The facies and faunal characters are typical of a ramp-like open shelf. The lack of reef-constructing organisms resulted in a gently dipping ramp morphology for the margin and slope. On the basis of facies analysis, three depositional sequences (third order) are defined.     

Evolution of sexual dimorphism and male dimorphism in the expression of beetle horns: phylogenetic evidence for modularity, evolutionary lability, and constraint

Beetle horns are enlarged outgrowths of the head or thorax that are used as weapons in contests over access to mates. Horn development is typically confined to males (sexual dimorphism) and often only to the largest males (male dimorphism). Both types of dimorphism result from endocrine threshold mechanisms that coordinate cell proliferation near the end of the larval period. Here, we map the presence/absence of each type of dimorphism onto a recent phylogeny for the genus Onthophagus (Coleoptera: Scarabaeidae) to explore how horn development has changed over time. Our results provide empirical support for several recent predictions regarding the evolutionary lability of developmental thresholds, including uncoupled evolution of alternative phenotypes and repeated fixation of phenotypes. We also report striking evidence of a possible developmental constraint. We show that male dimorphism and sexual dimorphism map together on the phylogeny; whenever small males have horns, females also have horns (and vice versa). We raise the possibility that correlated evolution of these two phenomena results from a shared element in their endocrine regulatory mechanisms rather than a history of common selection pressures. These results illustrate the type of insight that can be gained only from the integration of developmental and evolutionary perspectives.     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.