Tunicate

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Tunicates
Temporal range:
Ma[1]
(Possible Ediacaran record, 557 Ma[2][3])
Gold-mouth sea squirt (Polycarpa aurata)
Scientific classification Edit this classification
Domain: Eukaryota
Kingdom: Animalia
Phylum: Chordata
Clade: Olfactores
Subphylum: Tunicata
Lamarck, 1816[4][5]
Classes and unplaced genera[4][7]
Synonyms

Urochordata Lankester, 1877

A tunicate is a marine

vertebrates). The subphylum was at one time called Urochordata, and the term urochordates is still sometimes used for these animals. They are the only chordates that have lost their myomeric segmentation, with the possible exception of the 'seriation of the gill slits'.[8][9] However, doliolids still display segmentation of the muscle bands.[10]

Some tunicates live as solitary individuals, but others replicate by

sessile, immobile and permanently attached to rocks or other hard surfaces on the ocean floor. Thaliaceans (pyrosomes, doliolids, and salps) and larvaceans on the other hand, swim in the pelagic zone
of the sea as adults.

Various species of

sea squirts, sea pork, sea livers, or sea tulips
.

The earliest probable species of tunicate appears in the fossil record in the early

Cambrian period. Despite their simple appearance and very different adult form, their close relationship to the vertebrates is evidenced by the fact that during their mobile larval stage, they possess a notochord or stiffening rod and resemble a tadpole. Their name derives from their unique outer covering or "tunic", which is formed from proteins and carbohydrates, and acts as an exoskeleton
. In some species, it is thin, translucent, and gelatinous, while in others it is thick, tough, and stiff.

Taxonomy

Clavelina moluccensis, the bluebell tunicate
Botrylloides violaceus showing oral tentacles at openings of buccal siphons

About 3,000 species of tunicate exist in the world's oceans, living mostly in shallow water. The most numerous group is the

pelagic. Some are supported by a stalk, but most are attached directly to a substrate, which may be a rock, shell, coral, seaweed, mangrove root, dock, piling, or ship's hull. They are found in a range of solid or translucent colours and may resemble seeds, grapes, peaches, barrels, or bottles. One of the largest is a stalked sea tulip, Pyura pachydermatina, which can grow to be over 1 metre (3.3 ft) tall.[12]

The Tunicata were established by Jean-Baptiste Lamarck in 1816. In 1881, Francis Maitland Balfour introduced another name for the same group, "Urochorda", to emphasize the affinity of the group to other chordates.[13] No doubt largely because of his influence, various authors supported the term, either as such, or as the slightly older "Urochordata", but this usage is invalid because "Tunicata" has precedence, and grounds for superseding the name never existed. Accordingly, the current (formally correct) trend is to abandon the name Urochorda or Urochordata in favour of the original Tunicata, and the name Tunicata is almost invariably used in modern scientific works. It is accepted as valid by the World Register of Marine Species[14] but not by the Integrated Taxonomic Information System.[15]

Various common names are used for different species. Sea tulips are tunicates with colourful bodies supported on slender stalks.[16] Sea squirts are so named because of their habit of contracting their bodies sharply and squirting out water when disturbed.[17] Sea liver and sea pork get their names from the resemblance of their dead colonies to pieces of meat.[18]

Classification

Tunicates are more closely related to craniates (including hagfish, lampreys, and jawed vertebrates) than to lancelets, echinoderms, hemichordates, Xenoturbella or other invertebrates.[19][20][21]

The clade consisting of tunicates and vertebrates is called Olfactores.[22]

The Tunicata contain roughly 3,051 described species,[12] traditionally divided into these classes:

Members of the

paraphyletic status.[23][24][25] A close reationship between Thaliacea and Ascidiacea, with the former possibly emerging from the latter, had already been proposed since the early 20th century under the name of Acopa.[26]

The following cladogram is based on the 2018 phylogenomic study of Delsuc and colleagues.[25]

Tunicata

Fossil record

The star-shaped holes (Catellocaula vallata) in this Upper Ordovician bryozoan may represent a tunicate preserved by bioimmuration in the bryozoan skeleton.

Undisputed fossils of tunicates are rare. The best known and earliest unequivocally identified species is

Maotianshan Shale at Shankou village, Anning, near Kunming (South China).[27] There is also a common bioimmuration, (Catellocaula vallata), of a possible tunicate found in Upper Ordovician bryozoan skeletons of the upper midwestern United States.[28] A well-preserved Cambrian fossil, Megasiphon thylakos, shows that the tunicate basic body design had already been established 500 million years ago.[29]

Three enigmatic species were also found from the

Ausia fenestrata from the Nama Group of Namibia, the sac-like Yarnemia ascidiformis, and one from a second new Ausia-like genus from the Onega Peninsula of northern Russia, Burykhia hunti. Results of a new study have shown possible affinity of these Ediacaran organisms to the ascidians.[30][31] Ausia and Burykhia lived in shallow coastal waters slightly more than 555 to 548 million years ago, and are believed to be the oldest evidence of the chordate lineage of metazoans.[31] The Russian Precambrian fossil Yarnemia
is identified as a tunicate only tentatively, because its fossils are nowhere near as well-preserved as those of Ausia and Burykhia, so this identification has been questioned.

Fossils of tunicates are rare because their bodies decay soon after death, but in some tunicate families, microscopic spicules are present, which may be preserved as microfossils. These spicules have occasionally been found in Jurassic and later rocks, but, as few palaeontologists are familiar with them, they may have been mistaken for sponge spicules.[32]

In Permian and Triassic there were also forms with a calcareous exoskeleton. At first they were mistaken for corals.[33][34]

Hybridization studies

A multi-taxon

panarthropods and nematodes). This study was based on a quartet partitioning approach designed to reveal horizontal gene transfer events among metazoan phyla.[35]

Anatomy

Body form

Colonial tunicate with multiple openings in a single tunic.

Colonies of tunicates occur in a range of forms, and vary in the degree to which individual organisms, known as

zooids, integrate with one another. In the simplest systems, the individual animals are widely separated, but linked together by horizontal connections called stolons, which grow along the seabed. Other species have the zooids growing closer together in a tuft or clustered together and sharing a common base. The most advanced colonies involve the integration of the zooids into a common structure surrounded by the tunic. These may have separate buccal siphons and a single central atrial siphon and may be organized into larger systems, with hundreds of star-shaped units. Often, the zooids in a colony are tiny but very numerous, and the colonies can form large encrusting or mat-like patches.[12]

Body structure

By far the largest class of tunicates is the

tunicin, a variety of cellulose. The tunic is unique among invertebrate exoskeletons in that it can grow as the animal enlarges and does not need to be periodically shed. Inside the tunic is the body wall or mantle composed of connective tissue, muscle fibres, blood vessels, and nerves. Two openings are found in the body wall: the buccal siphon at the top through which water flows into the interior, and the atrial siphon on the ventral side through which it is expelled. A large pharynx occupies most of the interior of the body. It is a muscular tube linking the buccal opening with the rest of the gut. It has a ciliated groove known as an endostyle on its ventral surface, and this secretes a mucous net which collects food particles and is wound up on the dorsal side of the pharynx. The gullet, at the lower end of the pharynx, links it to a loop of gut which terminates near the atrial siphon. The walls of the pharynx are perforated by several bands of slits, known as stigmata, through which water escapes into the surrounding water-filled cavity, the atrium. This is criss-crossed by various rope-like mesenteries which extend from the mantle and provide support for the pharynx, preventing it from collapsing, and also hold up the other organs.[12]

The

salps are also small, under 4 cm (1.6 in) long, and found in the surface waters of both warm and cold seas. They also move by jet propulsion, and often form long chains by budding off new individuals.[12]

A third class, the

Larvacea (or Appendicularia), is the only group of tunicates to retain their chordate characteristics in the adult state, a product of extensive neoteny. The 70 species of larvaceans superficially resemble the tadpole larvae of amphibians, although the tail is at right angles to the body. The notochord is retained, and the animals, mostly under 1 cm long, are propelled by undulations of the tail. They secrete an external mucous net known as a house, which may completely surround them and is very efficient at trapping planktonic particles.[12]

Physiology and internal anatomy

Internal anatomy of a generalised tunicate
Section through the wall of an ascidian pyrosoma showing several zooids; (br) buccal siphon; (at) atrial siphon; (tp) test process; (br s) pharynx.

Like all other

epicardium, which is surrounded by the jelly-like mesenchyme
.

Ascidian tunicates begin life as a lecithotrophic (non-feeding) mobile

Appendicularia are pelagic, and the general larval form is kept throughout life. Also the class Thaliacea is pelagic throughout their lives and may have complex lifecycles. In this class a free living larval stage is absent: Doliolids and pyrosomatids are viviparous–lecithotrophic, and salpids are viviparous–matrotrophic. Only some species of doliolids still have a rudimentary tailed tadpole stage, which is never free-living and lacks a brain.[41][42][43]

Tunicates have a well-developed heart and circulatory system. The heart is a double U-shaped tube situated just below the gut. The blood vessels are simple connective tissue tubes, and their blood has several types of corpuscle. The blood may appear pale green, but this is not due to any respiratory pigments, and oxygen is transported dissolved in the plasma. Exact details of the circulatory system are unclear, but the gut, pharynx, gills, gonads, and nervous system seem to be arranged in series rather than in parallel, as happens in most other animals. Every few minutes, the heart stops beating and then restarts, pumping fluid in the reverse direction.[12]

Tunicate

vanadium-associated proteins in vacuoles in blood cells known as vanadocytes. Some tunicates can concentrate vanadium up to a level ten million times that of the surrounding seawater. It is stored in a +3 oxidation form that requires a pH of less than 2 for stability, and this is achieved by the vacuoles also containing sulfuric acid. The vanadocytes are later deposited just below the outer surface of the tunic, where their presence is thought to deter predation, although it is unclear whether this is due to the presence of the metal or low pH.[44] Other species of tunicates concentrate lithium, iron, niobium, and tantalum, which may serve a similar function.[12] Other tunicate species produce distasteful organic compounds as chemical defenses against predators.[45]

Tunicates lack the kidney-like metanephridial organs typical of deuterostomes. Most have no excretory structures, but rely on the diffusion of ammonia across their tissues to rid themselves of nitrogenous waste, though some have a simple excretory system. The typical renal organ is a mass of large clear-walled vesicles that occupy the rectal loop, and the structure has no duct. Each vesicle is a remnant of a part of the primitive coelom, and its cells extract nitrogenous waste matter from circulating blood. They accumulate the wastes inside the vesicles as urate crystals, and do not have any obvious means of disposing of the material during their lifetimes.[42]

Adult tunicates have a hollow cerebral ganglion, equivalent to a brain, and a hollow structure known as a neural gland. Both originate from the embryonic neural tube and are located between the two siphons. Nerves arise from the two ends of the ganglion; those from the anterior end innervate the buccal siphon and those from the posterior end supply the rest of the body, the atrial siphon, organs, gut and the musculature of the body wall. There are no sense organs but there are sensory cells on the siphons, the buccal tentacles and in the atrium.[12]

Tunicates are unusual among animals in that they produce a large fraction of their tunic and some other structures in the form of cellulose. The production in animals of cellulose is so unusual that at first some researchers denied its presence outside of plants, but the tunicates were later found to possess a functional cellulose synthesizing enzyme, encoded by a gene horizontally transferred from a bacterium.[46] When, in 1845, Carl Schmidt first announced the presence in the test of some ascidians of a substance very similar to cellulose, he called it "tunicine", but it is now recognized as cellulose rather than any alternative substance.[47][48][49]

Feeding

Clavelina robusta (black and white) and Pycnoclavella flava (orange) showing siphons.

Nearly all adult tunicates are

intestine, where absorption takes place, and the rectum, where undigested remains are formed into faecal pellets or strings. The anus opens into the dorsal or cloacal part of the peribranchial cavity near the atrial siphon. Here, the faeces are caught up by the constant stream of water which carries the waste to the exterior. The animal orientates itself to the current in such a way that the buccal siphon is always upstream and does not draw in contaminated water.[12]

Some ascidians that live on soft sediments are

Megalodicopia hians, are sit-and-wait predators, trapping tiny crustacea, nematodes, and other small invertebrates with the muscular lobes which surround their buccal siphons. Certain tropical species in the family Didemnidae have symbiotic green algae or cyanobacteria in their tunics, and one of these symbionts, Prochloron, is unique to tunicates. Excess photosynthetic products are assumed to be available to the host.[12]

Life cycle

Anatomy of a larval tunicate

Ascidians are almost all

ocellus, and a balancing organ, a statocyst.[50]

When sufficiently developed, the larva of the sessile species finds a suitable rock and cements itself in place. The larval form is not capable of feeding, though it may have a rudimentary digestive system,[50] and is only a dispersal mechanism. Many physical changes occur to the tunicate's body during metamorphosis, one of the most significant being the reduction of the cerebral ganglion, which controls movement and is the equivalent of the vertebrate brain. From this comes the common saying that the sea squirt "eats its own brain".[51] However, the adult does possess a cerebral ganglion adapted to lack of self-locomotion.[52] In the Thaliacea, the larval stage is rudimentary or suppressed, and the adults are pelagic (swimming or drifting in the open sea).[42] Colonial forms also increase the size of the colony by budding off new individuals to share the same tunic.[53]

Pyrosome colonies grow by budding off new zooids near the posterior end of the colony. Sexual reproduction starts within a zooid with an internally fertilized egg. This develops directly into an oozooid without any intervening larval form. This buds precociously to form four blastozooids which become detached in a single unit when the oozoid disintegrates. The atrial siphon of the oozoid becomes the exhalent siphon for the new, four-zooid colony.[12]

A 1901 comparison of frog tadpole and a tunicate larva.

Doliolids have a very complex life cycle that includes various zooids with different functions. The sexually reproducing members of the colony are known as gonozooids. Each one is a hermaphrodite with the eggs being fertilised by sperm from another individual. The gonozooid is viviparous, and at first, the developing embryo feeds on its yolk sac before being released into the sea as a free-swimming, tadpole-like larva. This undergoes metamorphosis in the water column into an oozooid. This is known as a "nurse" as it develops a tail of zooids produced by budding asexually. Some of these are known as trophozooids, have a nutritional function, and are arranged in lateral rows. Others are phorozooids, have a transport function, and are arranged in a single central row. Other zooids link to the phorozooids, which then detach themselves from the nurse. These zooids develop into gonozooids, and when these are mature, they separate from the phorozooids to live independently and start the cycle over again. Meanwhile, the phorozooids have served their purpose and disintegrate. The asexual phase in the lifecycle allows the doliolid to multiply very rapidly when conditions are favourable.[12]

Salps also have a complex lifecycle with an

sequential hermaphrodites. An egg in each is fertilized internally by a sperm from another colony. The egg develops in a brood sac inside the blastozooid and has a placental connection to the circulating blood of its "nurse". When it fills the blastozooid's body, it is released to start the independent life of an oozooid.[12]

Larvaceans only reproduce

gonochoric, and a larva resembles the tadpole larva of ascidians. Once the trunk is fully developed, the larva undergoes "tail shift", in which the tail moves from a rearward position to a ventral orientation and twists through 90° relative to the trunk. The larva consists of a small, fixed number of cells, and grows by enlargement of these rather than cell division. Development is very rapid and only takes seven hours for a zygote to develop into a house-building juvenile starting to feed.[12]

During embryonic development, tunicates exhibit

cell determination relatively late in development and cell cleavage is indeterminate. The genome evolution of amphioxus and vertebrates is also relatively slow.[54]

Promotion of out-crossing

Ciona intestinalis (class Ascidiacea) is a hermaphrodite that releases sperm and eggs into the surrounding seawater almost simultaneously. It is self-sterile, and thus has been used for studies on the mechanism of self-incompatibility.[55] Self/non-self-recognition molecules play a key role in the process of interaction between sperm and the vitelline coat of the egg. It appears that self/non-self recognition in ascidians such as C. intestinalis is mechanistically similar to self-incompatibility systems in flowering plants.[55] Self-incompatibility promotes out-crossing, and thus provides the adaptive advantage at each generation of the masking of deleterious recessive mutations (i.e. genetic complementation)[56] and the avoidance of inbreeding depression.

Botryllus schlosseri (class Ascidiacea) is a colonial tunicate, a member of the only group of chordates that are able to reproduce both sexually and asexually. B. schlosseri is a sequential (protogynous) hermaphrodite, and in a colony, eggs are ovulated about two days before the peak of sperm emission.[57] Thus self-fertilization is avoided, and cross-fertilization is favored. Although avoided, self-fertilization is still possible in B. schlosseri. Self-fertilized eggs develop with a substantially higher frequency of anomalies during cleavage than cross-fertilized eggs (23% vs. 1.6%).[57] Also a significantly lower percentage of larvae derived from self-fertilized eggs metamorphose, and the growth of the colonies derived from their metamorphosis is significantly lower. These findings suggest that self-fertilization gives rise to inbreeding depression associated with developmental deficits that are likely caused by expression of deleterious recessive mutations.[56]

A model tunicate

vertebrates.[19]

Invasive species

Over the past few decades, tunicates (notably of the genera Didemnum and Styela) have been invading coastal waters in many countries. The carpet tunicate (Didemnum vexillum) has taken over a 6.5 sq mi (17 km2) area of the seabed on the Georges Bank off the northeast coast of North America, covering stones, molluscs, and other stationary objects in a dense mat.[59] D. vexillum, Styela clava and Ciona savignyi have appeared and are thriving in Puget Sound and Hood Canal in the Pacific Northwest.[60]

Invasive tunicates usually arrive as

ballast water. Another possible means of introduction is on the shells of molluscs brought in for marine cultivation.[60] Current research indicates many tunicates previously thought to be indigenous to Europe and the Americas are, in fact, invaders. Some of these invasions may have occurred centuries or even millennia ago. In some areas, tunicates are proving to be a major threat to aquaculture operations.[61]

Use by humans

Medical uses

Tunicates contain a host of potentially useful chemical compounds, including:

  • Plitidepsin, a didemnin effective against various types of cancer; as of late January 2021 undergoing Phase III trials as a treatment for COVID-19[62]
  • Trabectedin, an FDA approved anticancer drug.

Tunicates are able to correct their own cellular abnormalities over a series of generations, and a similar regenerative process may be possible for humans. The mechanisms underlying the phenomenon may lead to insights about the potential of cells and tissues to be reprogrammed and to regenerate compromised human organs.[63][64][65]

As food

Sea squirts for sale at a market, Busan, South Korea

Various

cuisine of Chile, both raw and in seafood stews. In Japan and Korea, the sea pineapple (Halocynthia roretzi) is the main species eaten. It is cultivated on dangling cords made of palm fronds. In 1994, over 42,000 tons were produced, but since then, mass mortality events have occurred among the farmed sea squirts (the tunics becoming soft), and only 4,500 tons were produced in 2004.[66]

Other uses

The use of tunicates as a source of biofuel is being researched. The cellulose body wall can be broken down and converted into ethanol, and other parts of the animal are protein-rich and can be converted into fish feed. Culturing tunicates on a large scale may be possible and the economics of doing so are attractive. As tunicates have few predators, their removal from the sea may not have profound ecological impacts. Being sea-based, their production does not compete with food production as does the cultivation of land-based crops for biofuel projects.[67]

Some tunicates are used as model organisms. Ciona intestinalis and Ciona savignyi have been used for developmental studies. Both species' mitochondrial[68][69] and nuclear[70][71] genomes have been sequenced. The nuclear genome of the appendicularian Oikopleura dioica appears to be one of the smallest among metazoans[72] and this species has been used to study gene regulation and the evolution and development of chordates.[73]

See also

References

  1. S2CID 135091168
    .
  2. .
  3. .
  4. ^ a b Sanamyan, Karen (2013). "Tunicata". WoRMS. World Register of Marine Species. Retrieved 4 April 2013.
  5. S2CID 83266247
    .
  6. .
  7. ^ .
  8. .
  9. – via Google Books.
  10. S2CID 20806327. Retrieved 13 May 2023.{{cite journal}}: CS1 maint: multiple names: authors list (link
    )
  11. .
  12. ^ .
  13. ^ Foster, M. (ed.); Sedgwick, Adam (ed.); The Works of Francis Maitland Balfour. Vol. III. Memorial edition. Pub: Macmillan and co. 1885. May be downloaded from [1]
  14. ^ Tunicata World Register of Marine Species. Retrieved 2011-11-12.
  15. ^ Tunicata Lamarck, 1816 Integrated Taxonomic Information System. Retrieved 2017-03-30.
  16. ^ "Sea squirts and sea tulips". Australian Museum. Retrieved 25 September 2013.
  17. ^ "Sea squirt". Dictionary.com. Retrieved 25 September 2013.
  18. ^ "Sea pork, Aplidium stellatum". Smithsonian at Fort Pierce. Retrieved 25 September 2013.
  19. ^
    S2CID 4382758
    .
  20. .
  21. .
  22. ^ Jefferies, R. P. S. (1991) in Biological Asymmetry and Handedness (eds Bock, G. R.; Marsh, J.) pp. 94–127 (Wiley, Chichester).
  23. .
  24. .
  25. ^ .
  26. .
  27. .
  28. ^ Palmer, T. J.; Wilson, M. A. (1988). "Parasitism of Ordovician bryozoans and the origin of pseudoborings" (PDF). Palaeontology. 31: 939–949. Archived from the original (PDF) on 27 September 2013. Retrieved 7 April 2013.
  29. ^ Nagalwade, Vidya (7 July 2023). "A 500 million-year-old fossil reveals the amazing secrets of tunicate origins". Tech Explorist.
  30. ^ Vickers-Rich P. (2007). "Chapter 4. The Nama Fauna of Southern Africa". In: Fedonkin, M. A.; Gehling, J. G.; Grey, K.; Narbonne, G. M.; Vickers-Rich, P. "The Rise of Animals: Evolution and Diversification of the Kingdom Animalia", Johns Hopkins University Press. pp. 69–87
  31. ^ a b Fedonkin, M. A.; Vickers-Rich, P.; Swalla, B.; Trusler, P.; Hall, M. (2008). "A Neoproterozoic chordate with possible affinity to the ascidians: New fossil evidence from the Vendian of the White Sea, Russia and its evolutionary and ecological implications". HPF-07 Rise and fall of the Ediacaran (Vendian) biota. International Geological Congress - Oslo 2008.
  32. ^ "Introduction to the Urochordata". University of California Museum of Paleontology. Archived from the original on 21 April 2009. Retrieved 7 April 2013.
  33. ^ [https://www.cambridge.org/core/services/aop-cambridge-core/content/view/0FE5DCCCDFDD464B92DCA4AF68F36F2B/S0022336019001094a.pdf/rare_case_of_an_evolutionary_late_and_ephemeral_biomineralization_tunicates_with_composite_calcareous_skeletons.pdf A rare case of an evolutionary late and ephemeral biomineralization: tunicates with composite calcareous skeletons]
  34. S2CID 135456629
    – via CrossRef.
  35. .
  36. .
  37. .
  38. .
  39. .
  40. .
  41. .
  42. ^ – via archive.org.
  43. .
  44. .
  45. .
  46. .
  47. .
  48. .
  49. .
  50. ^ a b Cavanihac, Jean-Marie (2000). "Tunicates extraordinaire". Microscope UK. Retrieved 7 December 2011.
  51. .
  52. .
  53. ^ Parmentier, Jan (1998). "Botryllus: A colonial ascidian". Microscope UK. Retrieved 7 April 2013.
  54. S2CID 5549210
    .
  55. ^ .
  56. ^
    PMID 3324702. {{cite book}}: |journal= ignored (help
    )
  57. ^ .
  58. .
  59. ^ "Have You Seen This Tunicate?". NOAA Fisheries Service. 19 November 2004. Archived from the original on 9 January 2009. Retrieved 7 December 2011.
  60. ^ a b Dornfeld, Ann (1 May 2008). "Invasive Tunicates of Washington State". NPR. Archived from the original on 14 July 2014. Retrieved 6 April 2013.
  61. ^ "Marine Nuisance Species". Woods Hole Science Center. Retrieved 7 December 2011.
  62. ^ Johnson, Mark. "International team of scientists identifies new treatment for COVID-19 that appears to be far more effective than drugs in use now". Journal Sentinel.
  63. OCLC 233972733
    .
  64. ^ "Sea Squirt, Heal Thyself: Scientists Make Major Breakthrough in Regenerative Medicine". Sciencedaily.com. 24 April 2007. Retrieved 7 December 2011.
  65. S2CID 37526690
    .
  66. ^ "Sea squirt". Korea-US Aquaculture. Archived from the original on 2 March 2013. Retrieved 6 April 2013.
  67. ^ "Biofuel made from marine filter feeders? Tunicates usable as source of biofuels". Cleantechnica. 26 March 2013. Retrieved 6 April 2013.
  68. PMID 17640763
    .
  69. .
  70. S2CID 15987281.{{cite journal}}: CS1 maint: multiple names: authors list (link
    )
  71. .
  72. .
  73. .

External links