Invertebrate Zoology, 2017, 14(2): 217–225 © INVERTEBRATE ZOOLOGY, 2017

The ultrastructural organization of and their phylogenetic relationships

Ya.I. Zabotin

Kazan (Volga region) Federal University, Kazan, 420008, Russia. E-mail: Yaroslav_Zabotin@ rambler.ru

ABSTRACT: Acoela represent one of the most spectacular taxa in kingdom. Their phylogenetic position as well as a taxonomic rank widely varies in zoological literature from the order of flatworms to the separate phylum within the deuterostomes or the sister taxon of all other bilaterians. One of the reasons of the absence of consensus among morphologists and molecular biologists is the insufficient amount of fine structural data on Acoela. In the present work the new ultrastructural features of four species of Acoela (Archaphanostoma agile, Otocelis rubropunctata, Symsagittifera japonica and Amphiscolops sp.) from different families are described. Along with the archaic characteristics the new apomorphic morphological features of epidermis, body wall musculature and central syncytial paren- chyma of species studied were found. The cellular organization of acoels is characterized by the wide morphological diversity, however no significance of the secondarily simplifi- cation was found. The alternative views on phylogenetic affinities of Acoela are discussed, and the conclusion of progressive but not regressive evolution of this invertebrate group is proposed. How to cite this article: Zabotin Ya.I. 2017. The ultrastructural organization of Acoela and their phylogenetic relationships // Invert. Zool. Vol.14. No.2. P.217–225. doi: 10.15298/ invertzool.14.2.17

KEY WORDS: Acoela, morphology, ultrastructure, systematics, phylogeny.

Ультраструктурная организация бескишечных турбеллярий (Acoela) и их филогенетические отношения

Я.И. Заботин

Казанский (Приволжский) федеральный университет, Казань, 420008, Россия. E-mail: [email protected]

РЕЗЮМЕ: Бескишечные турбеллярии (Acoela) представляют собой один из самых необычных таксонов животного царства. Их филогенетическое положение и таксо- номический ранг варьируют в зоологической литературе от отряда плоских червей до отдельного типа вторичноротых или до сестринского таксона по отношению ко всем остальным Bilateria. Одной из причин отсутствия единого мнения у морфологов и молекулярных биологов является недостаток данных по ультратонкому строению Acoela. В работе приводятся новые данные по ультраструктуре четырех видов Acoela (Archaphanostoma agile, Otocelis rubropunctata, Symsagittifera japonica и Amphiscolops sp.) из разных семейств. Наряду с архаическими особенностями описаны новые 218 Ya.I. Zabotin

апоморфные особенности эпидермиса, мускулатуры стенки тела и центральной синцитиальной паренхимы у исследованных видов. Клеточная организация бески- шечных турбеллярий отличается высоким морфологическим разнообразием, однако свидетельств их вторичного упрощения обнаружено не было. Обсуждаются альтер- нативные взгляды на филогенетические связи Acoela и приводится заключение о прогрессивной, а не регрессивной эволюции этой группы беспозвоночных. Как цитировать эту статью: Zabotin Ya.I. 2017. The ultrastructural organization of Acoela and their phylogenetic relationships // Invert. Zool. Vol.14. No.2. P.217–225. doi: 10.15298/invertzool.14.2.17

КЛЮЧЕВЫЕ СЛОВА: Бескишечные турбеллярии (Acoela), морфология, ультра- структура, систематика, филогения.

Introduction matic phylum of worm-like invertebrates Xeno- turbellida; the newly-erected taxon Xenacoelo- The taxon Acoela includes small predomi- morpha is transferred back to the basis of the nantly marine (with just two known freshwater Bilaterian phylogenetic tree (Cannon et al., species as exceptions) worm-like invertebrates. 2016). So, up to date the phylogenetic position Up to date about 380 species of Acoela is of this problematic taxon still remains unclear. grouped into various number of families — One of the reasons of the absence of consensus from 9 (Jondelius et al., 2011) to 19 (Hooge, among morphologists and molecular biologists Tyler, 2005). Classical morphologists consid- is the insufficient amount of fine structural data ered them to be the most primitive members of on Acoela. So the aim of the present work is the the Bilateria (Ivanov, Mamkaev, 1973), or op- ultrastructural study of cellular organization of positely the secondarily simplified invertebrates, Acoela with phylogenetic implications. e.g. the paedomorphic descendants of advanced coelomate metazoans (Malakhov, 2009). Materials and methods The taxonomic status of Acoela is also vari- able in different sources. For a long time they Four species of Acoela were chosen for the have been classified within class Turbellaria, present study: Archaphanostoma agile (Jensen, subclass Archoophora (Ivanov, Mamkaev, 1878) (Isodiametridae), Otocelis rubropuncta- 1973). Ehlers (1985) proposed the systematic ta (Schmidt, 1852) (Otocelididae), Symsagit- revision of phylum Plathelminthes based on tifera japonica (Kato, 1951) (Sagittiferidae) ultrastructural features. He revealed the class and Amphiscolops sp. (Convolutidae). S. japon- Turbellaria to be paraphyletic taxon lacking ica (formerly known as Convoluta japonica) certain synapomorphies, and placed Acoela with and Amphiscolops sp. (described by Minegishi closely related order Nemertodermatida in (1965) without a specific name) have never subphylum Acoelomorpha. According to the been studied on electron-microscopic level be- results of 18S rDNA sequence, Ruiz-Trillo et al. fore. The specimens of A. agile were collected (1999) removed Acoelomorpha from the phy- on the sandy littoral of various islands of lum Plathelminthes and classified them as the Keretskii Archipelago (White Sea, Russia); the sister taxon to all other Bilateria; although Phil- other species were collected on sandy littoral ippe et al. (2011), based on multiple protein- and algae of Mukaishima Island (Hiroshima coding genes sequence, surprisingly placed them Prefecture, Japan). The entire were within a superphylum Deuterostomia. Finally, fixed in 1% glutaraldehyde on 0.1 M phosphate the most relevant molecular and morphological buffer. The preparation of the samples for the data join the Acoelomorpha with another enig- transmission electron microscopy (TEM) in- The ultrastructural organization of Acoela and their phylogenetic relationships 219 cluded post-fixation in 1% osmium tetraoxide dimental, while in other species they are well- on 0,1 M phosphate buffer, dehydration from developed. In the cytoplasm of epidermal cells 30% to absolute alcohol and acetone, embed- of three species (O. rubropunctata, S. japonica ding in epon resin. Ultrafine cuts were made and Amphiscolops sp.) below the ciliary rootlets using ultramicrotome Reichert-Jung, contrast- the multiple small transparent vacuoles (about 1 ed with uranil-acetate and lead citrate and inves- µm in diameter) are scattered (Fig. 1 B, C, v). tigated using TEM JEM 100 CX in the Depart- The body wall musculature of A. agile, O. ment of Zoology and General Biology of Kazan rubropunctata and S. japonica consists of three (Volga Region) Federal University (Kazan, layers of the smooth musculature — outer circu- Russia). lar, middle diagonal and inner longitudinal muscles; the degree of development of each Results layer differs depending on the species. In A. agile the diagonal muscles sometimes are irreg- Epidermis and body wall muscula- ularly arranged. In O. rubropunctata the diago- nal muscles are also rather poorly-differentiat- ture ed. Oppositely, the all three muscle layers of S. japonica are well-developed. The nuclei of the The single-layered epidermis of all four spe- myocytes reach 3 µm in diameter. cies studied is lacking a basal membrane and The body wall musculature of Amphiscol- weakly separated from parenchyma. The epi- ops sp. is well-developed and differs from the dermis of Archaphanostoma agile consists of above-mentioned species by unusual order of narrow cells 7–8 µm high and belongs to non- muscle layers: the diagonal muscles are located insunk type, characterized by location of cell below the circular and longitudinal ones (Fig. 1 nuclei upper to the musculature (Fig. 1 A). B). The outer circular layer is represented by the Otocelis rubropunctata also possess non-in- smooth muscles. The middle longitudinal and sunk epidermis; its cells reach 13 µm in height. inner diagonal layers consist of the cross-striat- Symsagittifera japonica and Amphiscolops sp. ed muscle fibers. The myofibrils are transver- possess a typical insunk epidermis (Fig. 1 B). sally crossed by the electron dense striations Each epithelial cell is divided by the muscula- resembling the Z-discs. The distance between ture on two parts – upper wide and flat terminal the striations (which can be considered as the plate, about 6 µm wide and 2 µm high and lower length of the sarcomere) is about 1.5 µm. basal part, containing nucleus about 4 µm in diameter. The epidermal cells are connected by Parenchyma the cell junctions — desmosomes — in the terminal parts. In all four studied species of Acoela paren- The external body surface of Acoela is cov- chyma is divided into peripheral and central ered by cilia, characterized by standard ax- zone (including the syncytium). The peripheral oneme formula 9+2. The kinetosome of each parenchyma of A. agile consists of three types of cilium possesses a single curved transversally- cells. There are elongated cells (5–7 µm in striated main rootlet up to 1 µm in length (Fig. 1 length) with large nucleus and electron trans- C–E, mr). Two very thin lateral rootlets go away parent cytoplasm (Fig. 2 A), the cells with dense from the curve of the main rootlet to the ends of cytoplasm and the small cells (3.5 µm in length) the rootlets of neighboring cilia (Fig. 1 C–E, lr). with high nuclear-cytoplasmic ratio. The cells Also a very short caudal rootlet is present (Fig. of central parenchyma are oval or amoeboid and 1 C–E, cr), from which the thin posterolateral reach 10 µm in length; electron transparent microtubules go away to the curves of the root- cytoplasm contains the numerous mitochon- lets of the neighboring cilia (Fig. 1 C–E, plm). In dria, dictyosomes of Golgi complex, channels A. agile the posterolateral microtubules are ru- of endoplasmic reticulum. The central syncy- 220 Ya.I. Zabotin

Fig. 1. The ultrastructure of epidermis and musculature of Acoela. A — non-insunk epidermis of Archaphanostoma agile; B — insunk epidermis and body wall musculature of Amphiscolops sp.; C — ciliary rootlets of epidermal cell of Otocelis rubropunctata; D — ciliary rootlets of epidermal cell of Amphiscolops sp.; E — schematic reconstruction of the ciliary rootlets of Acoela. Abbreviations: c — cilia; cm — circular muscles; cr — caudal rootlet; d — desmosome; dgm — diagonal muscles; e — epidermal cell; epr — endoplasmic reticulum; g — gland; lm — longitudinal muscles; lr — lateral rootlet; m — mitochondria; mr — main rootlet; n — nucleus; plm — posterolateral microtubules; pp — peripheral parenchyma; v — vacuole. Scale bar: 1 µm. Рис. 1. Ультраструктура эпидермиса и мускулатуры Acoela. A — непогруженный эпидермис Archaphanostoma agile; B — погруженный эпидермис и мускулатура стенки тела Amphiscolops sp.; The ultrastructural organization of Acoela and their phylogenetic relationships 221 tium is represented by loose conglomeration of to 4 µm in length) with highly condensed chro- cell organoids and digestive vacuoles. matin. The central parenchymal cells are oval or In O. rubropunctata the peripheral paren- round, reach 8 µm in diameter and contain the chyma includes four cell types: the oval cells solitary mitochondria and dictyosomes; these (up to 7 µm in diameter) with high nuclear- cells also form the central syncytium. cytoplasmatic ratio; the amoeboid cells (up to The parenchymal cells in all species studied 10 µm in diameter) with long processes; the are separated from each other by very thin layers irregular-shaped cells with the large nucleus; of extracellular matrix up to 0.25 µm wide. It is the small cells with unusual “inflated” perinu- filled with electron dense material and some- clear space. The cells of central parenchyma (13 times contains the small vesicles, probably the µm in diameter) are amoeboid with long pro- results of the autophagic processes. In the pa- cesses. The main volume of the central syncytial renchyma of S. japonica and Amphiscolops sp. mass is occupied by the large (up to 4 µm in the endosymbiotic green algae (zooxanthellae) diameter) digestive vacuoles and multilamellar were also observed. In some cases they were bodies (Fig. 2 B). found to be digested by the central syncytium. The peripheral parenchyma of S. japonica consists of three cell types. There are large cells Discussion (up to 7 µm in diameter) with the short process- es; the smaller cells (3.5 µm in diameter) with The epidermis of the four species of Acoela the large nucleus with wide “inflated” perinu- studied in the present work can be defined as clear space and the smallest cells (3 µm in non-insunk (in Archaphanostoma agile and diameter) with electron dense cytoplasm and Otocelis rubropunctata) and the insunk one (in small amount of organelles. The cells of central Symsagittifera japonica and Amphiscolops sp.). parenchyma reach 4 µm in diameter and possess Although Dorey (1965) originally classified the various shapes (oval, elongated or amoeboid). epidermis of O. rubropunctata as an insunk The central syncytium of S. japonica is sur- one, but according to our data, the nuclear parts rounded not only with parenchymal cells but of the cells never sink under the muscle layers. also with the muscle cells (Fig. 2 C). The latter The multiple vacuoles of unclear function found ones reach up to 4 µm in cross section and in epidermal cells in all species studied (except contain the well-distinguished myofibrils. In the A. agile) were also observed in Convoluta con- electron transparent central syncytial zone the voluta (Pedersen, 1964) and S. roscoffensis solitary organelles (e.g., cell nuclei) and the (formerly C. roscoffensis, Dorey, 1965). fragments of the cell membranes are loosely The ultrastructure of the ciliary rootlets of scattered. all species studied is typical for Acoela but The peripheral parenchyma of Amphiscol- never occurs in flatworms. The epidermal cell ops sp. is also composed of three cell types. cilium in most flatworms possesses two root- Among them there are oval cells (4 µm in diam- lets — the rostral and the caudal (sometimes eter) with the large nucleus and the cytoplasm of called ventral) and the basal foot of kinetosome the middle electron density; the larger cells (up to is reduced. Oppositely, in the Acoela these two 12 µm in diameter), amoeboid or star-shaped rootlets are fused into the single main rootlet with multiple processes; the elongated cells (up and additionally the pair of lateral rootlets ap-

C — корешки ресничек эпидермальной клетки Otocelis rubropunctata; D — корешки ресничек эпидермальной клетки Amphiscolops sp.; E — схематическая реконструкция корешков ресничек Acoela. Обозначения: c — реснички; cm — кольцевые мышцы; cr — задний корешок; d — десмосома; dgm — диагональные мышцы; e — клетка эпидермиса; epr — эндоплазматическая сеть; g — железа; lm — продольные мышцы; lr — латеральные корешки; m — митохондрия; mr — главный корешок; n — ядро; plm — постерола- теральные микротрубочки; pp — периферическая паренхима; v — вакуоль. Масштаб: 1 µm. 222 Ya.I. Zabotin

Fig. 2. The ultrastructure of parenchyma (A–C) and schematic reconstructions of body wall layers of Acoela (D–F). A — peripheral parenchyma (type I cells) of Archaphanostoma agile; B — central syncytium of Otocelis rubropunctata; C — central syncytium with muscular bound of Symsagittifera japonica; D — the plesiomorphic condition for Acoela: non-insunk epidermis, two-layered musculature and cellular parenchyma (exemplified by Paratomella rubra); E — the apomorphic condition for Acoela: insunk epidermis, three- layered musculature and central syncytium (in majority of acoel species); F — the autapomorphic condition for some derived families of Acoela: the muscular-bound central syncytium (exemplified by S. japonica). Abbreviations: c — cilia; cm — circular muscles; cp — central parenchyma; cs — central syncytium; dgm — diagonal muscles; dv — digestive vacuoles; e — epidermal cell; lm — longitudinal muscles; m — mitochondria; mbs — muscular bound of syncytium; mc — muscle cell; mlb — multilamellar body; n — nucleus; pp — peripheral parenchyma. Scale bar: 1 µm. The ultrastructural organization of Acoela and their phylogenetic relationships 223 pear; the caudal rootlet is homologous not to the The architectonics of body wall musculature above-mentioned caudal rootlet of other flat- is used as a taxonomic character in acoel phylo- worms but to the basal foot of the kinetosome genetics. In the basal families Diopisthoporidae, (see review in Ehlers, 1985; Raikova, 1991). Paratomellidae and Solenofilomorphidae the The rootlet system of Nemertodermatida differs diagonal muscles are absent and the whole body by the absence of the lateral rootlets (Tyler, musculature includes only the outer circular and Rieger, 1977) and in Xenoturbellida these two the inner longitudinal ones (Hooge, Tyler, 2006). rootlets are convergent but not fused (Lundin, The higher acoel families possessing the cross- 1998). The structure of ciliary rootlets and the like arrangement of diagonal musculature and pattern of the ending of the epidermal cilia the U-shaped muscles, surrounding the mouth axoneme represent the synapomorphies of the opening are now grouped within a taxon Cruci- Acoelomorpha (Ehlers, 1985). The monophyly musculata (Jondelius et al., 2011). of is proposed on the basis of All acoel species investigated in the present the peculiarities of epidermal cilia and their work possess the typical number and order of rootlets (Lundin, 1998) and the modern molec- the muscle layers for the crucimusculates ex- ular data (Cannon et al., 2016). However the cept the Amphiscolops sp. This species is char- Xenoturbellida significantly differ from Acoela acterized by the inner position of the diagonal by the development of epithelized intestine, the muscles. It should be mentioned that similar intraepidermal diffuse nervous system without unusual order of muscle layers (circular, longi- a cerebral ganglion (Raikova et al., 2000), the tudinal, diagonal) was shown for another con- absence of copulatory organs, the external in- volutid Oxyposthia praedator (Ivanov, 1952). semination and the primitive uniflagellate sper- Moreover in Amphiscolops sp. the cross-striat- matozoa (Obst et al., 2011). Thus, to our opinion, ed musculature was unexpectedly found. Pre- the phylogenetic relationships of Acoelomorpha viously the cross-striated muscles were de- and Xenoturbellida still remain debatable. scribed only in light-optical level in O. sacha- Raikova (1991) supposed that the plesio- linensis, Ox. praedator (Ivanov, 1952) and C. morphic condition of the epidermal ciliary root- convoluta (Westblad, 1948), but the ultrastruc- lets of the common ancestor of Acoelomorpha tural investigations of the latter species by and Plathelminthes (or probably of Bilateria in Pedersen (1964) didn’t confirm that. Raikova general) was the presence of the separated rostral (1989) also define the acoel musculature as a and caudal rootlets and the well-developed basal smooth one, but in some cases she describes foot of the kinetosome. Concerning that, the the dense striations of myofibrils as the homo- fusion of both these rootlets into a single main logues of the Z-discs. Concerning that, to our rootlet and the independent origin of the caudal opinion, the origin of cross-striated muscula- rootlet from the basal foot should be considered ture within the Acoela is highly probable, espe- as an apomorphy for the whole taxon Acoela. cially in the most advanced acoel family Convo-

Рис. 2. Ультраструктура паренхимы (A–C) и схематические реконструкции стенки тела Acoela (D– F). A — периферическая паренхима (клетки I типа) Archaphanostoma agile; B — центральный синцитий Otocelis rubropunctata; C — центральный синцитий с мышечной обкладкой Symsagittifera japonica; D — плезиоморфное состояние Acoela: непогруженный эпидермис, двухслойная мускула- тура и клеточная паренхима (на примере Paratomella rubra); E — апоморфное состояние Acoela: погруженный эпидермис, трехслойная мускулатура и центральный синцитий (у большинства видов Acoela); F — аутапоморфное состояние некоторых семейств Acoela: центральный синцитий с мышечной обкладкой (на примере S. japonica). Обозначения: c — реснички; cm — кольцевые мышцы; cp — центральная паренхима; cs — центральный синцитий; dgm — диагональные мышцы; dv — пищеварительные вакуоли; e — клетка эпидермиса; lm — продольные мышцы; m — митохондрия; mbs — мышечная обкладка синцития; mc — мышечная клетка; mlb — мультиламеллярное тело; n — ядро; pp — периферическая паренхима. Масштаб: 1 µm. 224 Ya.I. Zabotin lutidae (Jondelius et al., 2011) the Amphiscol- system are described. Among them there is the ops sp. belongs to. formation of the solitary non-epithelized diges- Summarizing all this data, we can conclude tive cavity in Actinoposthia beklemischevi. that the non-insunk epidermis, the absence of According to the ultrastructural data (Raikova, diagonal muscles and the presence of the exclu- 1987), this cavity is surrounded by the tempo- sively smooth musculature, widely distributed rary syncytial structures and is filled with the in the archaic acoels, represent the plesiomor- cellular derivates and the membrane-bound ves- phic condition for the whole taxon (Fig. 2 D). In icles. The typical parenchymal cells are separat- a contrast, in the advanced acoel families the ed from syncytium by the muscle bound, pro- non-insunk epidermis is usually replaced by the tecting them from its phagocytic activity (Raik- insunk one; the body wall musculature differen- ova, 1987). The muscle-bound cavity of A. tiates into the three layers and (at least in some beklemischevi and the central syncytium de- species such as Amphiscolops sp.) the cross- scribed here for S. japonica look similar in the striated muscles appear. These ultrastructural electron micrographs so it is possible that they peculiarities can be considered the progressive both represent the different physiological con- apomorphic condition for Acoela (Fig. 2 E). ditions of the same structure. The muscle-bound The literary and original ultrastructural data syncytium is also supposed to assume the sup- reveal the high diversity of cells in parenchyma portive function along with the traditional di- of Acoela. E.g., the parenchyma of C. convoluta gestive one. These cases exemplify the high consists of three cell types in the peripheral zone degree of integration between the parenchyma and two types in the central one (Pedersen, and musculature within the acoel organism. It 1964). Mamkaev and Markosova (1979) de- should be notified that Actinoposthiidae and scribed four cell types in the parenchyma of Ox. Convolutidae (including the members of the praedator: the large amoeboid cells (also mod- former Sagittiferidae family, to authors’ opin- ified into the syncytium), the poorly-differenti- ion) nowadays occupy the derived positions on ated cells (neoblasts), the reserve cells and the the phylogenetic tree of Acoela (Jondelius et al., glandular cells. Smith (1981) described in the 2011). parenchyma of Convoluta sp. the central syncy- The evolutionary-morphological analysis of tium and three types of so-called “wrapping literary and original data shows that plesiomor- cells”. The combination of cell types and their phic condition of digestive system of Acoela is quantitative composition in the parenchyma can represented by the exclusively cellular con- be considered as the specific characters. struction of the parenchyma (exemplified by P. The syncytial structure of digestive system rubra, Fig. 2 D). With increasing of body size is undoubtedly one of the modular features of and respectively the size of ingested prey the cellular organization of Acoela. In one of the archaic mode of phagocytic cellular digestion most primitive acoel species Paratomella rubra became non-effective. The intestine-less Acoe- the syncytial structures are absent and the paren- la have “solved” this problem by unique way – chyma consists only of cells (Smith, Tyler, by the syncytial transformation of the central 1985). According to the modern phylogenetic parenchyma. Thus the development of the cen- analysis, the family Paratomellidae represents tral syncytium is considered to be the apomor- the sister taxon to all other acoels except the phic condition for this taxon (Fig. 2 E). Finally, Diopisthoporidae (Jondelius et al., 2011). In the the origin of the muscular bound of syncytium in majority of acoel species the central syncytium some representatives of Acoela (e.g., A. bekle- is always present permanently or at least tempo- mischevi and S. japonica) can probably be con- rarily during the process of food digestion (Smith, sidered the autapomorphic condition for the Tyler, 1985; Raikova, 1987). derived families of Acoela (Fig. 2 F). Finally, in the some species of Acoela the The general conclusions can be summarized examples of deeper specialization of digestive as follows. The present study revealed the new The ultrastructural organization of Acoela and their phylogenetic relationships 225 ultrastructural features of Acoela which can be Mamkaev Yu.V., Markosova T.G. 1979. [Electron micro- identified as apomorphic characters, such as the scopic investigation of parenchyma of acoel turbellar- ians] // Trudy Zoologicheskogo Instituta AN SSSR. origin of cross-striated musculature in Amphis- Vol.84. P.7–12 [in Russian with English summary]. colops sp. and the central syncytium with the Minegishi H. 1965. [Acoela-Turbellaria] // New Illustrat- muscular bound in S. japonica. So the cellular ed Encyclopedia of the Fauna of Japan, Part I. Tokyo: organization of Acoela is characterized by the Hokuryu-kan Publ. P.311–312 [in Japanese]. Obst M., Nakano H., Bourlat S.J., Thorndyke M.C., Tel- wider morphological diversity than previously ford M.J., Nyengaard J.R., Funch P. 2011. Spermato- supposed, however no significance of the sec- zoon ultrastructure of Xenoturbella bocki (Westblad ondarily simplification was found. According 1949) // Acta Zoologica (Stockholm). Vol.92. P.109– 115. to the literary and original data, the Acoela are Pedersen K.J. 1964. 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