Molecular evidence that Sclerolinum brattstromi is closely related to vestimentiferans, not to frenulate pogonophorans (Siboglinidae, Annelida)

Siboglinids, previously referred to as pogonophorans, have typically been divided into two groups, frenulates and vestimentiferans. Adults of these marine protostome worms lack a functional gut and harbor endosymbiotic bacteria. Frenulates usually live in deep, sedimented reducing environments, and vestimentiferans inhabit hydrothermal vents and sulfide-rich hydrocarbon seeps. Taxonomic literature has often treated frenulates and vestimentiferans as sister taxa. Sclerolinum has traditionally been thought to be a basal siboglinid that was originally regarded as a frenulate and later as a third lineage of siboglinids, Monilifera. Evidence from the 18S nuclear rDNA gene and the 16S mitochondrial rDNA gene presented here shows that Sclerolinum is the sister clade to vestimentiferans although it lacks the characteristic morphology (i.e., a vestimentum). The rDNA data confirm the contention that Sclerolinum is different from frenulates, and further supports the idea that siboglinid evolution has been driven by a trend toward increased habitat specialization. The evidence now available indicates that vestimentiferans lack the molecular diversity expected of a group that has been argued to have Silurian or possibly Cambrian origins.     

Endosymbionts of Siboglinum fiordicum and the phylogeny of bacterial endosymbionts in Siboglinidae (Annelida)

Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known-vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of gamma-proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.     

Endosymbioses between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)

Siboglinid tubeworms do not have a mouth or gut and live in obligate associations with bacterial endosymbionts. Little is currently known about the phylogeny of frenulate and moniliferan siboglinids and their symbionts. In this study, we investigated the symbioses of two co-occurring siboglinid species from a methane emitting mud volcano in the Arctic Ocean (Haakon Mosby Mud Volcano, HMMV): Oligobrachia haakonmosbiensis (Frenulata) and Sclerolinum contortum (Monilifera). Comparative sequence analysis of the host-specific 18S and the symbiont-specific 16S rRNA genes of S. contortum showed that the close phylogenetic relationship of this host to vestimentiferan siboglinids was mirrored in the close relationship of its symbionts to the sulfur-oxidizing gammaproteobacterial symbionts of vestimentiferans. A similar congruence between host and symbiont phylogeny was observed in O. haakonmosbiensis: both this host and its symbionts were most closely related to the frenulate siboglinid O. mashikoi and its gammaproteobacterial symbiont. The symbiont sequences from O. haakonmosbiensis and O. mashikoi formed a clade unaffiliated with known methane- or sulfur-oxidizing bacteria. Fluorescence in situ hybridization indicated that the dominant bacterial phylotypes originated from endosymbionts residing inside the host trophosome. In both S. contortum and O. haakonmosbiensis, characteristic genes for autotrophy (cbbLM) and sulfur oxidation (aprA) were present, while genes diagnostic for methanotrophy were not detected. The molecular data suggest that both HMMV tubeworm species harbour chemoautotrophic sulfur-oxidizing symbionts. In S. contortum, average stable carbon isotope values of fatty acids and cholesterol of -43 per thousand were highly negative for a sulfur oxidizing symbiosis, but can be explained by a (13)C-depleted CO(2) source at HMMV. In O. haakonmosbiensis, stable carbon isotope values of fatty acids and cholesterol of -70 per thousand are difficult to reconcile with our current knowledge of isotope signatures for chemoautotrophic processes.     

The Osedax trophosome: organization and ultrastructure

The polychaete family Siboglinidae, which is currently construed as comprising the Frenulata, Monilifera (composed of Sclerolinum), Vestimentifera, and Osedax, has become known for its specialized symbiont-housing organ called the trophosome. This organ replaced the digestive system of the worms and is located in the elongated trunk region in Frenulata, Sclerolinum, and Vestimentifera. Currently two types of trophosomes have been described: in the taxa Frenulata and Sclerolinum the bacteriocytes originate from endoderm, and in Vestimentifera they originate from mesoderm. In Osedax, a trophosome was described as lacking (Rouse et al., 2004), but bacteriocytes are located in Osedax's characteristic root tissue. Here, we argue for a consistent name for the symbiont-housing tissue, namely trophosome, as in other siboglinids. In this study we provide morphological evidence that in Osedax the bacteriocytes are derived from somatic mesoderm. We show that the trophosome in Osedax is an apolar tissue composed of bacteriocytes and nonsymbiotic cells. As in vestimentiferans, a specific cell cycle was identified; however, in this case it is directed from the posterior to the anterior end of the worms instead of from the center toward the periphery. Comparison of all siboglinid trophosomes and re-evaluation of their body regions allows us to discuss whether the trophosomes are homologous and to hypothesize about the organization of the last common ancestor of Siboglinidae.     

Siboglinid evolution shaped by habitat preference and sulfide tolerance

Siboglinids are tube-dwelling annelids that inhabit marine reducing habitats such as anoxic mud bottoms, seeps and hydrothermal vents. As adults, they lack a functional digestive system and rely on chemoautotrophic microbial endosymbionts. Based on morphological analyses, Siboglinidae form a clade with the Sabellariidae, Serpulidae and Sabellidae within the Annelida. The sister group to this clade is the Oweniidae. Three subgroups constitute the Siboglinidae: Frenulata typically inhabit anoxic sediments, Sclerolinium (a.k.a., Monilifera) live on decaying organic matter or reduced sediments and Vestimentifera are mostly found at hydrocarbon seeps and hydrothermal vents. Recent studies suggest that Sclerolinum is the sister group to the Vestimentifera. Within the Vestimentifera, the species inhabiting bare-rock hydrothermal vents represent a derived clade. The seep-inhabiting genus Lamellibrachia forms a basal branch within the Vestimentifera. Trends in siboglinid evolution are most notable with regard to the level of sulfide tolerance and type of substrate. Basal groups inhabit soft substrate with only slightly elevated sulfide levels, whereas more derived species colonize hard substrate and tolerate elevated temperatures and high levels of sulfide. The type of substrate correlates with tube morphology and the function of the opisthosome. The role of the symbionts in habitat selection needs further investigation.     

Phylogeny of Vestimentifera (Siboglinidae, Annelida) inferred from morphology

Vestimentifera, formerly considered a phylum, are here included in the annelid clade Siboglinidae which also encompasses Frenulata and Sclerolinum . All Siboglinidae inhabit reducing habitats, mostly in the deep sea. Vestimentifera are known from hydrothermal vents and cold seeps. Cladistic analyses of vestimentiferan relationships are performed on three levels: (1) among the vestimentiferan species, (2) among the reconstructed ancestral vestimentiferan and other siboglinids and (3) on the level of the families included in the annelidan clade Sabellida. The monophyly of vestimentiferans is confirmed in all analyses. A group of exclusively vent-inhabiting species forms a derived monophyletic clade. The sister group to the vent clade is the Escarpia complex. Lamellibrachia appears to be paraphyletic. Except for the paraphyly of Lamellibrachia , the reconstructed pattern agrees with the molecular phylogeny based on cytochrome c oxidase subunit I. Ancient ridge systems can be invoked to explain modern day geographical distributions. The Pacific Kula Ridge that spanned the Pacific in an east–west direction during the Early Tertiary, may have been a pathway for the ancestor of the vent clade to reach the eastern Pacific. The biogeography is consistent with the recent divergence of Vestimentifera as inferred from molecular data. The reconstructed phylogeny of the Siboglinidae supports the monophyly of the Frenulata and within those, the Thecanephria and Athecanephria. In contrast to molecular and other morphological analyses, Sclerolinum appears as the sister group to the Frenulata. The family level analysis supports the sister group relationship of the Siboglinidae to a clade formed by Sabellariidae, Sabellidae and Serpulidae. Hypothesized homologies of the vestimentiferan obturaculum and vestimentum to structures in related taxa need further investigation.     

A cladistic analysis of Siboglinidae Caullery, 1914 (Polychaeta, Annelida): formerly the phyla Pogonophora and Vestimentifera

It has been proposed in recent years that the phyla Pogonophora and Vestimentifera are a derived clade of polychaete annelids. It has also been proposed that if this clade belongs among polychaetes, then the taxon name Pogonophora is misleading and should revert to a name first formulated for the group, Siboglinidae Caullery, 1914. This recommendation is adopted in this paper, and a cladistic study using terminals of 'generic' rank in the former Pogonophora (including Vestimentifera) is undertaken. The purpose of this is to assess which taxon names should now be used for clades within Siboglinidae, and to provide a revised taxonomy, based on phylogenetic principles. Another major aim is to assess the position of the vestimentiferan clade within Siboglinidae. The results show that Vestimentifera is the sister group to Sclerolinum, and this clade is then sister group to Frenulata, i.e. the remaining Siboglinidae. The results suggest that all taxa within Siboglinidae that are not genera or species are redundant, except for the following: Siboglinidae is defined as the first polychaete, and all its descendants, to have an gut occluded by expanded endoderm filled with chemoautotrophic bacteria, as seen in the holotype of Riftia pachyptila Jones, 1981. Monilifera can be defined based on apomorphy-based system such that it is the first siboglinid, and all its descendants, to have rings of chaetae (uncini) in the opisthosoma, as seen in the holotype of Sclerolinum magdalenae Southward, 1972. Vestimentifera can be denned as the first siboglinid and all its descendants to have a vestimentum as seen in the holotype of Riftia pachyptia. Frenulata is defined as the siboglinid, and all its descendants, to have a mid-trunk girdle, as seen in the holotype of Siboglinum weberi Caullery, 1914. The taxa of generic rank are not defined here since their monophyly was not investigated.     

Microbial diversity in Frenulata (Siboglinidae,Polychaeta) species from mud volcanoes in the Gulf of Cadiz (NE Atlantic)

Frenulates are a group of gutless marine annelids belonging to the Siboglinidae that are nutritionally dependent upon endosymbiotic bacteria. We have characterized the bacteria associated with several frenulate species from mud volcanoes in the Gulf of Cadiz by PCR-DGGE of bacterial 16S rRNA genes, coupled with analysis of 16S rRNA gene libraries. In addition to the primary symbiont, bacterial consortia (microflora) were found in all species analysed. Phylogenetic analyses indicate that the primary symbiont in most cases belongs to the Gammaproteobacteria and were related to thiotrophic and methanotrophic symbionts from other marine invertebrates, whereas members of the microflora were related to multiple bacterial phyla. This is the first molecular evidence of methanotrophic bacteria in at least one frenulate species. In addition, the occurrence of the same bacterial phylotype in different Frenulata species, from different depths and mud volcanoes suggests that there is no selection for specific symbionts and corroborates environmental acquisition as previously proposed for this group of siboglinids.     

Unsegmented annelids? Possible origins of four lophotrochozoan worm taxa

In traditional classification schemes, the Annelida consists of the Polychaeta and the Clitellata (the latter including the Oligochaeta and Hirudinida). However, recent analyses suggest that annelids are much more diverse than traditionally believed, and that polychaetes are paraphyletic. Specifically, some lesser-known taxa (previously regarded as separate phyla) appear to fall within the annelid radiation. Abundant molecular, developmental, and morphological data show that the Siboglinidae, which includes the formerly recognized Pogonophora and Vestimentifera, are derived annelids; recent data from the Elongation Factor-1α (EF-1α) gene also suggest that echiurids are of annelid ancestry. Further, the phylogenetic origins of two other lesser-known groups of marine worms, the Myzostomida and Sipuncula, have recently been called into question. Whereas some authors advocate annelid affinities, others argue that these taxa do not fall within the annelid radiation. With advances in our understanding of annelid phylogeny, our perceptions of body plan evolution within the Metazoa are changing. The evolution of segmentation probably is more plastic than traditionally believed. However, as our understanding of organismal evolution is being revised, we are also forced to reconsider the specific characters being examined. Should segmentation be considered a developmental process or an ontological endpoint?     

The reproductive system of Osedax (Annelida,Siboglinidae): ovary structure,sperm ultrastructure,and fertilization mode

Osedax is a genus of siboglinid annelids in which the females live on dead vertebrate bones on the seafloor. These females have a posterior end that lies within the bone and contains the ovarian tissue, as well as the “roots” involved with bone degradation and nutrition. The males are microscopic and live as “harems” in the lumen of the gelatinous tube that surrounds the female trunk, well away from the ovary. Females are known to spawn fertilized primary oocytes, suggesting internal fertilization. However, little is known about sperm transfer, sperm storage, or the location of fertilization, and the morphology of the female reproductive system has not been described and compared with the reproductive systems of other siboglinids. A 3D‐reconstruction of the ovisac of Osedax showed ovarian tissue with multiple lobes and mature oocytes stored in a “uterus” before being released through the single oviduct. The oviduct emerges as a gonopore on the trunk and travels along the trunk to finally open to the seawater as a thin cylindrical tube among the crown of palps. Light and transmission electron microscopy of mature Osedax sperm revealed elongate heads consisting of a nucleus with helical grooves occupied by mitochondria. In contrast to other Siboglinidae, Osedax sperm are not packaged into spermatophores or spermatozeugmata, and Osedax females lack a discrete region for sperm storage. Transmission electron microscopy and fluorescence microscopy allowed detection of sperm associated with ovarian tissue of the female ovisac of four different Osedax species. This provides the first evidence for the site of internal fertilization in Osedax. A heart body was found in the circulatory system, as seen in other siboglinids and some other annelids. The possible presence of nephridia in the anterior ovisac region was also documented. These morphological features provide new insights for comparing the regionalization of Osedax females in relation to other siboglinids.     

Morphology, microanatomy and sequence data of Sclerolinum contortum (Siboglindae, Annelida) of the Gulf of Mexico

Sclerolinum is a small genus of Siboglinidae (Annelida) living in an obligate mutualistic association with thiotrophic bacteria as adults. Its taxonomic position, based on morphology, has been controversial; however, molecular data point to a sister taxa relationship with vestimentiferans. 16S rRNA gene sequencing and comparative morphology revealed that the studied population from deep-sea hydrocarbon seeps of the Gulf of Mexico belongs to Sclerolinum contortum known from the Arctic Sea. Since no anatomical and microanatomical studies have been published yet, we conducted such a study on S. contortum using serial sectioning and light and transmission electron microscopy. We show that the Sclerolinum body, divided into a head, trunk, and opisthosoma, is very similar to that of the vestimentiferans, and therefore we propose that the body regions are homologous in both taxa.     

New morphological evidence supports congruent phylogenies and Gondwana vicariance for palaeognathous birds

There has been little agreement on the phylogeny of palaeognathous birds, with major differences amongst and between results from morphological and molecular data. Two recently published phylogenies using nuclear and mitochondrial DNA have substantial agreement in overall topology, with the ostrich as sister group of all other extant palaeognaths and a kiwi‐emu‐cassowary clade. Here I report a morphological phylogeny based mainly on new characters from the tongue apparatus and cranial osteology, with a theoretical ancestor as outgroup. A new interpretation of the evolution of the avian palate is included. This phylogeny is very similar to these recent molecular results; this is the first report of such congruence, and offers a credible basis for understanding the evolution of this clade. This phylogeny is fully consistent with a Gondwana vicariance model of evolution. Dates attributed from known geological events place the first extant radiation (ostrich) in the mid‐Cretaceous, and offer a means of calibration of future molecular clock investigations. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 959–983.     

Close phylogenetic relationship between vestimentifera (tube worms) and annelida revealed by the amino acid sequence of elongation factor-lα

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-l (EF-l) gene (939–1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-l, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.Correspondence to: S. Kojima     

Hyalinoecia artifex: Field notes on a charismatic and abundant epifaunal polychaete on the US Atlantic continental margin

Hyalinoecia artifex is an abundant species on the US Atlantic margin, although little is known about its behavior, ecology, or reproduction. We provide field notes and high‐resolution in situ imagery of this species observed and collected in 2013 and 2015. Hyalinoecia artifex occurred primarily on soft sediment at a density of 10.9±0.95 individuals m^?2 (mean±standard error). Although they were found adjacent to mussel beds associated with methane seeps, worms were negatively associated with authigenic carbonate and other epibenthic megafauna in the study area. Quill worms moved across the sediment by extending their heads from the tubes and dragging the tubes forward. Stable isotope analysis indicated that H. artifex is a secondary consumer. Most individuals collected in 2015 were ripe, with mature oocytes up to 400 μm. Oocytes were yolky, yellow in color and positively buoyant; no brooding was observed.     

Organization and microanatomy of the Sclerolinum contortum trophosome (Polychaeta, Siboglinidae)

The trophosome-an organ especially evolved to accommodate symbiotic bacteria-is a key character of the polychaete family Siboglinidae. Astonishingly, the trophosomes vary in organization and origin between the different siboglinid taxa. The trophosome of the small genus Sclerolinum was nearly unknown until now. Here we investigated the trophosome of S. contortum from the Gulf of Mexico, using light and electron microscopy. We show that this organ derives from the visceral mesoderm and propose that the trophosome of the sister clade Vestimentifera and Sclerolinum is a homologous character. Like that of juvenile vestimentiferans, the trophosome of Sclerolinum trophosome is simply organized. This study reveals that the Sclerolinum trophosome exhibits two regions that differ in the organization of host tissue and the size and shape of the symbionts. We suggest that a specific cell cycle within the symbiont-housing organ is directed along the longitudinal body axis, with a region of proliferation anteriorly and a region of degradation posteriorly. Using Raman microspectroscopy we demonstrate that the endosymbionts of S. contortum from the Gulf of Mexico contain sulfur vesicles, and we argue for a chemoautotrophic sulfur-oxidizing metabolism.     

Evolution in the pentastomids

The Pentastomida, or tongue worms, form an aberrant group of annulated endoparasites little known to most parasitologists and veterinarians. Although there is no doubt of their affiliation to arthropods, textbooks often rank the Pentastomida as a separate phylum. Riley's many publications on the group have 'promoted it from the realm of the completely unknown to the semi-obscure', but still much research is needed on their basic biology, as little is known about their biochemistry, ecology or their evolutionary relationships. This review by Rolf Haugerud is intended to inspire curiosity about the Pentastomida by discussing their evolution in relation to their phylogeny, host relationships, geographic distribution, transmission and impact.     

Phylogeny of the water strider genus Rheumatobates (Heteroptera: Gerridae)

The genus Rheumatobates comprises thirty‐seven species and subspecies of New World water striders belonging to subfamily Rhagadotarsinae. Among species, males vary dramatically in the degree and nature of modifications of the antennae, three pairs of legs and abdominal and genital segments. Characters describing this modification have traditionally been used to differentiate and group species. The general assumption has been that modified species belong to one group and unmodified species to another. These two ‘species groups’ are subdivided into ‘subgroups’, but little effort has been made to resolve relationships among them. We conduct the first numerical cladistic analysis of Rheumatobates using a data set comprised of 102 characters, primarily describing modification of male external morphology. To address concerns about the inclusion of characters to be optimized on the phylogeny, characters describing modification of antennae and hind legs were included and then excluded in separate analyses. A preferred phylogeny was chosen from the four equally parsimonious cladograms found after successive reweighting of characters. There was good resolution at all levels of the phylogeny. Most of the major clades and terminal relationships were moderately to strongly supported, whereas the basal relationships were less well supported. The general assumption that unmodified and modified species form two monophyletic groups was not supported. However, traditionally recognized ‘subgroups’ within the modified species group were largely upheld. The analysis also suggested several major clades and relationships among these clades that were not previously recognized. The exclusion of characters describing modification of antennae and hind legs did not change the resolved major clades of the reconstructed phylogeny.     

Spermatozoa and sperm aggregates in the vestimentiferan Lamellibrachia luymesi compared with those of Riftia pachyptila (Polychaeta: Siboglinidae: Vestimentifera)

The spermatozoa and the sperm bundles of the vestimentiferans Riftia pachyptila and Lamellibrachia luymesi (Annelida: Siboglinidae) were studied using several microscopical techniques (transmission and scanning electron microscopy, and confocal microscopy) and compared with some other annelid sperm. The spermatozoa and sperm bundles of both species show a similar structure, but they differ in the dimensions of the components of individual cells and in the number of spermatozoa forming each sperm bundle. The spermatozoa of R. pachyptila and L. luymesi are filiform cells composed, in sequence, by an acrosome in the form of a thread-like helical vesicle, an elongated coiled nucleus surrounded by two helical mitochondria, and a long flagellum. In the spermatozoa of both species, the apical portion of the nucleus is completely devoid of chromatin and is delimited by a thickened nuclear envelope with a fibrillar appearance. Both species have sperm bundles that resemble buds, having a calyx-like portion formed by the helical heads, and a stalk-like portion formed by the tightly packed flagella. A parsimony analysis based on spermatozoal characters showed monophyly of the Siboglinidae and the Vestimentifera. We propose a new set of autapomorphies characterizing vestimentiferan spermatozoa. Our analysis suggests that spermatozoal characters are useful to the understanding of the phylogeny of the group.     

Revision of the Bicyclus sciathis species group (Lepidoptera: Nymphalidae) with descriptions of four new species and corrected distributional records

In this paper we present a thorough revision of the sciathis species group of the butterfly genus Bicyclus (Kirby). Type materials are discussed and in several cases lectotypes are assigned to specimens from original type series. Four new, and morphologically distinct, species are described (B. elishiae Brattström sp.n. , B. heathi Brattström sp.n. , B. sigiussidorum Brattström sp.n. and B. subtilisurae Brattström sp.n. ), along with a comprehensive molecular phylogeny that includes exemplar taxa of all currently recognized species. We also investigate the types of all previously synonymized taxa and in the process invalidate the name B. ewondo Libert. This was done after finding the previously missing holotype of B. makomensis (Strand), which clearly belongs to the same species and thereby gives the older name priority. The phylogeny showed that some distinctly different species were surprisingly closely related, suggesting a high rate of morphological evolution in parts of the sciathis group. The distributional records for the group are updated after investigating over 1700 specimens kept in a range of museum collections. Many species previously thought to be broadly sympatric were found to have much more restricted ranges, with the previous overestimations probably based on misidentified specimens. The higher level of allopatry now established will make identification of many morphologically similar species easier. The fact that species often have smaller ranges than previously known, meaning that the level of endemism for African butterflies is likely to be higher than current estimates, has important implications for conservation management. An identification key for males of all 13 currently recognized species in the species group is included. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:837A9D4C‐779A‐4497‐8176‐7151D409DFA5 .