Origin of Vertebrates/Chapter III
CHAPTER III
THE EVIDENCE OF THE SKELETON
The bony and cartilaginous skeleton considered, not the notochord.—Nature of the earliest cartilaginous skeleton.—The mesosomatic skeleton of Ammocœtes; its topographical arrangement, its structure, its origin in muco-cartilage.—The prosomatic skeleton of Ammocœtes; the trabeculæ and parachordals, their structure, their origin in white fibrous tissue.—The mesosomatic skeleton of Limulus compared with that of Ammocœtes; similarity of position, of structure, of origin in muco-cartilage.—The prosomatic skeleton of Limulus; the entosternite or plastron compared with the trabeculæ of Ammocœtes; similarity of position, of structure, of origin in fibrous tissue.—Summary.
The explanation of the two optic diverticula given in the last chapter accounts in the same harmonious manner for every other part of the tube around which the central nervous system of the vertebrate has been grouped. The tube conforms in all respects to the simple epithelial tube which formed the alimentary canal of the ancient type of marine arthropods such as were dominant in the seas when the vertebrates first appeared. The whole evidence so far is so uniform and points so strongly in the direction of the origin of vertebrates from these ancient arthropods, as to make it an imperative duty to proceed further and to compare one by one the other parts of the central nervous system, together with their outgoing nerves in the two groups of animals.
Before proceeding to do this, it is advisable first to consider the question of the origin of the vertebrate skeletal tissues, for this is the second of the great difficulties in the way of deriving vertebrates from arthropods, the one skeleton being an endo-skeleton composed of cartilage and bone, and the other an exo-skeleton composed of chitin. Here is a problem of a totally different kind to that we have just been considering, but of so fundamental a character that it must, if possible, be solved before passing on to the consideration of the cranial nerves and the organs they supply.
Is there any evidence which makes it possible to conceive the method by which the vertebrate skeleton may have arisen from the skeletal tissues of an arthropod? By the vertebrate skeleton I mean the bony and cartilaginous structures which form the backbone, the cranio-facial skeleton, the pectoral and pelvic girdles, and the bones of the limbs. I do not include the notochord in these skeletal tissues, because there is not the slightest evidence that the notochord played any part in the formation of these structures; the notochordal tissue is something sui generis, and never gives rise to cartilage or bone. The notochord happens to lie in the middle line of the body and is very conspicuous in the lowest vertebrate; with the development of the backbone the notochord becomes obliterated more and more, until at last it is visible in the higher vertebrates only in the embryo; but that obliteration is the result of the encroachment of the growing bone-masses, not the cause of their growth. Although, then, the notochord may in a sense be spoken of as the original supporting axial rod of the vertebrate, it is so different to the rest of the endo-skeleton, has so little to do with it, that the consideration of its origin is a thing apart, and must be treated by itself without reference to the origin of the cartilaginous and bony skeleton.
The Commencement of the Bony Skeleton in the Vertebrate.
What is the teaching of the vertebrate? What evidence is there as to the origin of the bony skeleton in the vertebrate phylum itself?
The axial bony skeleton of the higher Mammalia consists of two parts, (1) the vertebral column with its attached bony parts, and (2) the cranio-facial skeleton. Of these two parts, the bony tissue of the first arises in the embryo from cartilage, of the second partly from cartilage, partly from membrane.
In strict accordance with their embryonic origin is their phylogenetic origin: as we pass from the higher vertebrates to the lower these structures can be traced back to a cartilaginous and membranous condition, so that, as Parker has shown, the cranio-facial bony skeleton of the higher vertebrates can be derived directly from a non-bony cartilaginous skeleton, such as is seen in Petromyzon and the cartilaginous fishes.
Balfour, in his "Comparative Embryology," states that the primitive cartilaginous cranium is always composed of the following parts:-
1. A pair of cartilaginous plates on each side of the cephalic section of the notochord known as the parachordals (pa.ch., Fig. 49; iv., Fig. 48). These plates, together with the notochord (ch.) enclosed between them, form a floor for the hind and mid-brain.
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Fig. 48.—Embryo Pig, two-thirds of an inch long (from Parker), Elements of Skull seen from below.
ch., notochord; iv., parachordals; au., auditory capsule; py., pituitary body; tr., trabecula; ctr., trabecular cornu; pn., pre-nasal cartilage; ppg., palato-pterygoid tract; mn., mandibular arch; th.h., first branchial arch; VII.-XII., cranial nerves.
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Fig. 49.—Head of Embryo Dog-fish (from Parker), Basal View of Cranium from above.
ol., olfactory sacs; au., auditory capsule; py., pituitary body; pa.ch., parachordal cartilage; tr., trabecula; inf., infundibulum; pt.s., pituitary space; e., eye.
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2. A pair of bars forming the floor for the fore-brain, known as the trabeculæ (tr). These bars are continued forward from the parachordals. They meet posteriorly and embrace the front end of the notochord, and after separating for some distance bend in again in such a way as to enclose a space—the pituitary space (pt.s.). In front of this space they remain in contact, and generally unite. They extend forward into the nasal region (pn.).
3. The cartilaginous capsules of the sense organs. Of these the auditory (au.) and the olfactory capsules (ol.) unite more or less intimately with the cranial walls; while the optic capsules, forming the usually cartilaginous sclerotics, remain distinct.
The parachordals and notochord form together the basilar plate, which forms the floor for that section of the brain belonging to the primitive postoral part of the head, and its extent corresponds roughly to that of the basioccipital of the adult skull.
The trabeculæ, so far as their mere anatomical relations are concerned, play the same part in forming the floor for the front cerebral vesicle as do the parachordals for the mid- and hind-brain. They differ, however, from the parachordals in one important feature, viz. that except at their hinder end they do not embrace the notochord. The notochord always terminates at the infundibulum, and the trabeculæ always enclose a pituitary space, in which lies the infundibulum (inf.) and the pituitary body (py.).
In the majority of the lower forms the trabeculæ arise quite independently of the parachordals, though the two sets of elements soon unite.
The trabeculæ are usually somewhat lyre-shaped, meeting in front and behind, and leaving a large pituitary space between their middle parts. Into this space the whole base of the fore-brain primitively projects, but the space itself gradually becomes narrowed until it usually contains only the pituitary body.
The trabecular floor of the brain does not long remain simple. Its sides grow vertically upwards, forming a lateral wall for the brain, in which in the higher types, two regions may be distinguished, viz. an alisphenoidal region behind, growing out from what is known as the basisphenoidal region of the primitive trabeculæ, and an orbito-sphenoidal region in front, growing out from the presphenoidal region of the trabeculæ. These plates form at first a continuous lateral wall of the cranium. The cartilaginous walls which grow up from the trabecular floor of the cranium generally extend upwards so as to form a roof, though almost always an imperfect roof, for the cranial cavity.
The basi-cranial cartilaginous skeleton reduces itself always into trabeculæ and parachordals with olfactory and auditory cartilaginous capsules.
In addition, a branchial skeleton exists, which consists of a series of bars known as the branchial bars, so situated as to afford support to the successive branchial pouches. An anterior arch known as the mandibular arch (Fig. 50, Mn.), placed in front of the hyo-mandibular cleft, and a second arch, known as the hyoid arch (Hy.), placed in front of the hyo-branchial cleft, are developed in all types; the succeeding arches are known as the true branchial arches (Br.), and are only fully developed in the Ichthyopsida. In all cases of jaw-bearing (gnathostomatous) vertebrates the first arch has become a supporting skeleton for the mouth (Fig. 51), and in the higher vertebrates in combination with the second or hyoid arch takes part in the formation of the ear-bones.
Fig. 50.—Head of Embryo Dog-fish, eleven lines long. (From Parker.)
Fig. 51.—Skull of Adult Dog-fish, Side View. (From Parker.)
cr., cranium; Br., branchial arches; Mn. + Hy., mandibular and hyoid arches.
The true branchial arches persist, to a certain extent, in the Amphibia, and become still more degenerated in the Amniota (reptiles, birds, and mammals) in correlation with the total disappearance of a branchial respiration at all periods of their life. Their remnants become more or less important parts of the hyoid bone, and are employed solely in support of the tongue.
In no single animal is there any evidence that the foremost arch, the mandibular, is a true branchial arch. As low down as the Elasmobranchs it becomes divided into two elements which form respectively the upper and lower jaws; the hyoid arch, on the other hand, although it has altered its form and acquired the secondary function of supporting the mandibular arch, still retains its respiratory function.
The evidence afforded by the mode of formation of the skeletal tissues of vertebrates down to the Elasmobranchs indicates that the primitive cranial skeleton arose from two paired basal cartilages, the parachordals and trabeculæ, to which were attached respectively cartilaginous cases enclosing the organs of hearing and smell. In addition, the branchial portion of the cranial region was provided with cartilaginous bars arranged serially for the support of the branchiæ, with the exception of the foremost, the mandibular bar, which formed supporting tissues for the mouth—the upper and lower jaws.
Just as in past times the spinal nerves and the segments they supplied were supposed to represent the type on which the original vertebrate was built, so also the spinal vertebræ afforded the type of the segmented skeleton, and the anatomists of those days strove hard to resolve the cranio-facial skeleton into a series of modified vertebræ. Owing especially to the labours of Huxley, who showed that the segmentation in the head-region was essentially a segmentation due to the presence of branchial bars, this conception was finally laid to rest and nowadays it is admitted to be hopeless to resolve the cranium into vertebral segments. Still, however, the vertebrate is a segmented animal and its segmented nature is visible in the cranial region, so far as the skeletal tissues are concerned, in the shape of the series of branchial and visceral bars.
To this segmentation the name of 'branchiomeric' has been given, while that due to the presence of vertebræ is called 'mesomeric.'
As we have seen, the internal bony skeleton of the vertebrate commences as a cartilaginous and membranous skeleton. For this reason the preservation of such skeletons is impossible in the fossil form, unless the cartilage has become impregnated with lime salts, so that there is but little hope of ever obtaining traces of such structures in the fossils of the Silurian age either among the vertebrate or invertebrate remains. Fortunately for this investigation there are still living on the earth two representatives of that age; on the invertebrate side Limulus, and on the vertebrate side Ammocœtes.
The Elasmobranchs represent the most primitive of the gnathostomatous vertebrates. Below them come the Agnatha, known as the cyclostomatous fishes or Marsipobranchii, the lampreys (Petromyzon) and the hag-fishes (Myxine).
The skeleton of Petromyzon (Fig. 52) consists of a cranio-facial skeleton composed of a cartilaginous unsegmented cranium, with the basal trabeculæ and parachordals and a series of branchial and visceral cartilaginous bars forming the so-called branchial basket-work; to these must be added auditory and nasal capsules. In contradistinction to this elaborate cranio-facial skeleton, the spinal vertebral skeleton is represented only by segmentally arranged small pieces of cartilage formed in the connective tissue dissepiments between segmented sheets of body-muscles (myotomes).
Fig. 52.—Skeleton of Petromyzon. (From Parker.)
na., nasal capsule; au., auditory capsule; nc., notochord.
But Petromyzon is derived from Ammocœtes by a remarkable process of transformation, and a most important part of that transformation is the formation of new cartilaginous structures. Thus we see that in Ammocœtes there is no sign of a cartilaginous vertebral column; at transformation the rudimentary vertebræ of Petromyzon are formed. In Ammocœtes the brain-case is a simple fibrous membranous covering; at transformation this becomes cartilaginous. In Ammocœtes there are no cartilaginous structures corresponding to the sub-ocular arches; these are all formed at transformation. It follows, that we can trace back the bony skeleton of the vertebrate head to the skeleton of Ammocœtes, and we may therefore conclude that the primitive cartilaginous skeleton of the vertebrate consisted of the following structures (Fig. 53, B), viz. the branchial bars forming a basket-work, the trabeculæ and parachordals, the auditory and nasal capsules—a clear proof that the cranial skeleton is older than the spinal. Of these structures the branchial bars are the only evidently segmented parts.
Fig. 53.—Comparison of Cartilaginous Skeleton of Limulus and Ammocœtes.
A, Diagram of cartilaginous skeleton of Limulus. Soft cartilage, entapophysial ligaments, deep black; branchial bars simply hatched; hard cartilage, lateral trabeculæ of entosternite, netted; Ph., position of pharynx.
B, Diagram of cartilaginous skeleton of Ammocœtes. Soft cartilage, sub-chordal cartilaginous bands, deep black; branchial basket-work (first formed part), simply hatched; hard cartilage, cranio-facial skeleton, trabeculæ, parachordals and auditory capsules, netted; Inf., position of tube of infundibulum (old œsophagus).
The Soft Cartilage of the Branchial Skeleton of Ammocœtes.
The study of Ammocœtes gives yet another clue to the nature of the earliest skeleton, for these two marked groups of cartilage—the branchial and basi-cranial—are characterized by a difference in structure as well as a difference in topographical position. J. Müller was the first to point out that these two sets of cartilages differ in appearance and constitution, and he gave to them the name of yellow and grey cartilage. Parker has described them fully under the terms soft and hard cartilage, terms which Schaffer has also used, and I shall also make use of them here. The whole of the branchial cartilaginous skeleton is composed of soft cartilage, while the basi-cranial skeleton, consisting of trabeculæ, parachordals, and auditory capsule, is composed of hard cartilage, the only soft cartilage in this region being that which forms the nasal capsule, not represented in Fig. 53, B.
These two groups of cartilage arise independently, so that at first the basi-cranial system is quite separate from the branchial, and only late in the history of the animal is a junction effected between the branchial system and the trabeculæ and parachordals, an initial separation which is especially striking when we consider that in this animal all the cartilaginous structures of any one system are continuous: there is no sign of anything in the nature of joints.
Of these two main groups, the branchial cartilages are formed first in the embryo, a fact which suggests that they are the most primitive of the vertebrate cartilages, and that, therefore, the first true formation of cartilage in the invertebrate ancestor may be looked for in the shape of bars supporting the branchial mechanism. The evidence of the origin of the cartilaginous structures in Ammocœtes is given by Shipley in the following words:—
"The branchial bases are the first part of the skeleton to appear. They arise about the 24th day as straight bars of cartilage, lying external and slightly posterior to the branchial vessel.
"The first traces of the basi-cranial skeleton appear on the 30th day as two rods of cartilage—the trabeculæ."
Our attention must, in the first place, be directed to this branchial basket-work of Ammocœtes.
Underlying the skin of Ammocœtes in the branchial region is situated the sheet of longitudinal body-muscles, divided into a series of segments or myotomes, which forms the somatic muscles so characteristic of all fishes. This muscular sheet is depicted on the left-hand side of Fig. 54. It does not extend over the lower lip or over that part in the middle line where the thyroid gland is situated. In these parts a sheet of peculiar tissue known by the name of muco-cartilage lies immediately under the skin, covering over the thyroid gland and lower lip. The somatic muscular sheet with the superjacent skin can be stripped off very easily owing to the vascularity and looseness of the tissue immediately underlying it. When this is done the branchial basket-work comes beautifully into view as is seen on the right-hand side of Fig. 54. It forms a cage within which the branchiæ and their muscles lie entirely concealed.
This is the great characteristic of this most primitive form of the branchial cartilaginous bars and distinguishes it from the branchial bars of other higher fishes, in that it forms a system of cartilages which lie external to the branchiæ—an extra-branchial system.
This branchial basket-work is simpler in Ammocœtes than in Petromyzon, and its actual starting-point consists of a main transverse bar corresponding to each branchial segment; from this transverse bar the system of longitudinal bars by which the basket-work is formed has sprung. These transverse bars arise from a cartilaginous longitudinal rod, situated close against the notochord on each side. These rods may be called the subchordal cartilaginous bands (Fig. 53), and, according to the observations of Schneider and others, each subchordal band does not form at first a continuous cartilaginous rod, but the cartilage is conspicuous only at the places where the transverse bars arise. In the youngest Ammocœtes examined by Schaffer, he could find no absolute discontinuity of the cartilage except between the first two transverse bars, but he says that the thinning between the transverse bars was so marked as to make it highly probable that at an earlier stage there was discontinuity. The whole system of branchial bars and subchordal rods is at first absolutely disconnected from the cranial system of trabeculæ and parachordals, and only later do the two systems join.
Fig. 54.—Ventral View of Head Region of Ammocœtes.
Th., thyroid gland; M., lower lip, with its muscles.
These observations on Ammocœtes lead most definitely to the conclusion that the starting-point of the whole cartilaginous skeleton of the vertebrate consisted of a series of transverse cartilaginous bars, for the purpose of supporting branchial segments; these were connected with two axial longitudinal cartilaginous rods, which at first contained cartilage only near the places of junction of the branchial bars. This system may be called the mesosomatic skeleton, as it is entirely confined to the branchial or mesosomatic region.
In addition to this primitive cartilaginous framework, which was formed for the support of the mesosomatic or respiratory segments, but at a slightly later period in the phylogenetic history, a separate cartilaginous system was formed for the support of the prosomatic segments, viz. the trabeculæ and parachordals with the auditory capsules: a system which was at first entirely separated from the mesosomatic, and, as we shall see, is more advanced in structure than the branchial system. Later still, the story is completed at the time of transformation to Petromyzon by the formation of the simple cartilaginous skull and the rudimentary vertebræ, the structure of which is also of a more advanced type.
The Structure of the Soft Branchial Cartilage.
Having considered the topographical position of the primitive branchial cartilaginous skeleton, we may now inquire, What was its structure and how was it formed?
In the higher vertebrates various forms of cartilage are described, viz. hyaline, fibro-cartilage, elastic cartilage, and parenchymatous cartilage. Of these, the parenchymatous cartilage is looked upon as the most primitive form, because it preserves without modification the characters of embryonic cartilage.
Embryology, then, would lead to the belief that the earliest form of cartilage in the vertebrate kingdom ought to be of this type, viz. large cells, each of which is enclosed in a simple capsule, so that the capsules of the cells form the whole of the matrix, and thus form a simple homogeneous honeycomb-structure, in the alveoli of which the cartilage-cells lie singly. If, then, the branchial cartilages of Ammocœtes are, as has just been argued, the representatives of the cartilaginous skeleton of the primitive vertebrate, it is reasonable to suppose that they should resemble in structure this embryonic cartilage. Such is undoubtedly the case: all observers who have described the branchial basket-work of Ammocœtes or Petromyzon have been struck with the extremely primitive character of the cartilage, and the last observer (Schaffer) describes it as composed of thin walls of homogeneous material, in which there are no lines of separation, which form a simple honeycomb-structure, in the alveoli of which the separate cells lie singly. These branchial cartilages are each surrounded by a layer of perichondrium, and in Fig. 55, A, I give a picture of a section of a portion of one of the bars.
Fig. 55.—A, Branchial Cartilage of Ammocœtes, stained with Thionin. B, Branchial Cartilage of Limulus, stained with Thionin.
Hence we see that structurally as well as topographically the branchial bars of Ammocœtes justify their claim to be considered as the origin of the vertebrate cartilaginous framework.
On the Structure of the Muco-cartilage in Ammocœtes.
We can, however, go further than this, and ask how this cartilage itself is formed in Ammocœtes? The answer is most definite, most instructive and suggestive, for in all cases this particular kind of cartilage is formed from, or at all events in, a peculiar fibrous tissue, which was called by Schneider "Schleim-Knorpel," or muco-cartilage, a tissue which is distinguishable from other connective tissues, not only by its structural peculiarities, but also by its strong affinity for all dyes which differentiate mucoid or chondro-mucoid substances.
This muco-cartilage is thus described by Schneider:—The perichondrium in Ammocœtes is not confined to the true cartilaginous structures, but extends itself in the form of thin plates in definite directions. Between these plates of perichondrium a peculiar tissue (Fig. 56)—the muco-cartilage—exists, consisting of fibrillæ, whose direction is mainly at right angles to the planes of the perichondrial plates, with star-shaped cells in among them, and with the spaces between the fibrillæ filled up with a semi-fluid mass.
From this tissue all the primitive cartilages which resemble the branchial bars are formed, either by the invasion of chondroblasts from the surrounding perichondrium, or by the proliferation and encapsulation of the cells of the muco-cartilage itself.
Fig. 56.—Section of Muco-cartilage from Dorsal Head-plate of Ammocœtes.
This very distinctive tissue—the muco-cartilage—is of very great importance in all questions of the origin of the skeletal tissues. In all descriptions of the skeletal tissues it has been practically disregarded until recent years when, besides my own observations, its distribution has been mapped out by Schaffer. Thus Parker, in his well-known description of the skeleton of the marsipobranch fishes, does not even mention its existence. Its importance is shown by its absolute disappearance at transformation and its non-occurrence in any of the higher vertebrates. It is entirely confined to the head-region, and its distribution there is most suggestive, for, as will be described fully later on, it forms a skeleton which both in structure and position resembles very closely the head-shields of cephalaspidian fishes. At the present part of my argument its more immediate interest lies in the method of tracing this tissue. For this purpose I made use of the micro-chemical reaction of thionin, a dye which, as shown by Hoyer, stains all mucin-containing substances a bright purple. Schaffer made use of a corresponding basophil stain, hæmalum. When stained with thionin, the matrix, or ground-substance of the branchial cartilages as well as the matrix or semi-fluid substance in which the fibrils of the muco-cartilaginous cells are embedded take on a deep purple colour, while the fibrous material of the cranial walls and other connective tissue strands, such as the perichondrium, are coloured light blue. Muco-cartilage, then, may be described as a peculiar form of connective tissue which differs from other connective tissue not only in its appearance but in its chemical composition, for unlike white fibrous tissue it contains a large amount of mucin, and this tissue is the forerunner of the earliest cartilaginous vertebrate skeleton, the branchial bars of Ammocœtes.
The conclusions to which we are led by the study of the structure, position, and mode of origin of these primitive cartilages of Ammocœtes may be thus summed up:—
1. The immediate ancestor of the vertebrate must have possessed a peculiar fibrous tissue—the ground-substance of which stained deep purple with thionin—in which cartilage arose.
2. The cartilage so formed was not like hyaline cartilage, but resembled in a striking manner parenchymatous cartilage.
3. This cartilage was situated partly in two axial longitudinal bands, partly as transverse bars, which supported the branchial apparatus.
The Prosomatic or Basi-cranial Skeleton of Ammocœtes.
Before searching for any evidence of a similar tissue in any invertebrate group, it is advisable to consider the other portion of the cartilaginous skeleton of Ammocœtes, which consists of the trabeculæ, parachordals and auditory capsules—the basi-cranial skeleton—and is composed of hard, not soft cartilage.
This basi-cranial skeleton represented in Fig. 53, B, is confined to the region of the notochord, the cranial walls being composed entirely of a white fibrous membrane. It is separated at first entirely from the sub-chordal portion of the branchial basket-work, and is composed of a foremost part, the trabeculæ (Tr.), and of a hindermost part, the parachordals (Pr.ch.), which are characterized by the attachment on each side of the large auditory capsule (Au.). In Ammocœtes the trabecular bars are continuous with the parachordals, the junction being marked by a small lateral projection on each side, which at transformation is seen to play an important part in the formation of the sub-ocular arch. The trabecular bar lies close against the notochord on each side up to its termination; it then bends away from the middle line and curves round until it meets its fellow on the opposite side, thus forming, as it were, the head of a racquet of which the notochord forms the splice in the handle. The strings of the racquet are represented by a thin membrane, in the centre of which the position of the infundibulum (Inf.) of the brain can be clearly seen. In an earlier stage of Ammocœtes the two trabecular horns do not meet, but are separated by connective tissue, which afterwards becomes cartilaginous.
As far, then, as the topography of this basi-cranial skeleton is concerned, the striking points are—the shape of the trabecular portion, diverging as it does around the infundibulum, and the presence on the parachordal portion of the two large auditory capsules.
These two points indicate, on the hypothesis that infundibulum and œsophagus are convertible terms, that two supporting structures of a cartilaginous nature must have existed in the ancestor of the vertebrate, the first of which surrounded the œsophagus, and the second was in connection with its auditory apparatus.
Structure of the Hard Cartilages.
The structure of this hard cartilage of the trabeculæ and auditory capsules resembles that of the soft, in so far that it consists of large cells with a comparatively small amount of intercellular substance. Schaffer, who has described it lately, considers that it is a nearer approach to hyaline cartilage than the soft, but yet cannot be called hyaline cartilage in the usual sense of the term. Its peculiarities and its differences from the soft are especially well seen by its staining reactions. I have myself been particularly struck with the effect of picrocarmine or combined hæmatoxylin and picric acid staining (Fig. 57). In the case of the soft cartilage the capsular substance stains respectively a brilliant red or blue, while that of the hard cartilage is coloured a deep yellow, so that the junction between the parachordals and the branchial cartilages is beautifully marked out. Then, again, with thionin, which gives so marked a reaction in the case of the soft cartilage, the hard cartilage of the auditory capsule is not stained at all, and in the trabeculæ the deep purple colour is confined to the mucoid cement-substance between the capsules, just as Schaffer has stated. The same kinds of reactions have been described by Schaffer: thus by double staining with hæmalum-eosin the hard cartilage stains red, the soft blue; and he points out that even with over-staining by hæmalum the auditory capsule remains colourless, just as I have noticed with thionin. He infers, precisely as I have done from the thionin reaction, that chondro-mucoid, which is so marked a constituent of the soft cartilage and of the muco-cartilage, is absent or present in but slight quantities in the hard cartilage. Similarly, he points out that double staining with tropœolin-methyl-violet stains the hard cartilage a bright orange colour, and the soft cartilage a violet.
The evidence, then, shows clearly that a marked chemical difference exists between these two cartilages, which may be expressed by saying that the one contains very largely a basophil substance, which we may speak of as belonging to the class of chondro-mucoid substances, while the other contains mainly an oxyphil substance, probably a chondro-gelatine substance.
We may perhaps go further and attribute this difference of composition to a difference of origin; for whereas the soft cartilage is invariably formed in a special tissue, the muco-cartilage, which shows by its reaction how largely it is composed of a mucoid substance, the hard cartilage is certainly, in the case of the cartilage of the cranium where its origin has been clearly made out, formed in the membranous tissue of the cranium of Ammocœtes—i.e. in a tissue which stains light blue with thionin, and contains a gelatinous rather than a mucoid substratum.
The best opportunity of finding out the mode of origin of the hard cartilage is afforded at the time of transformation, when so much of this kind of cartilage is formed anew. Unfortunately, it is very difficult to obtain the early transformation stages, consequently we cannot be said to possess any really exhaustive and definite account of how the new cartilages are formed. Bujor, Kaensche, and Schaffer all profess to give a more or less definite account of their formation, and the one striking impression left on the mind of the reader is how their descriptions vary. In one point only are they agreed, and in that I also agree with them, viz. the manner in which the new cranial walls are formed. Schaffer describes the process as the invasion of chondroblasts into the homogeneous fibrous tissue of the cranial walls. Such chondroblasts not only form the cartilaginous framework, but also assimilate the fibrous tissue which they invade, so that finally all that remains of the original fibrous matrix in which the cartilage was formed are these lines of cement-substance between the groups of cartilage cells, which, containing some basophil material, are marked out, as already mentioned (Fig. 57).
We may therefore conclude, from the investigation of Ammocœtes, that the front part of the basi-cranial skeleton arose as two trabecular bars, to which muscles were attached, situated bilaterally with respect to the central nervous system. These bars were composed of tendinous material with a gelatinous rather than a mucoid substratum, in which nests of cartilage-cells were formed, the cartilaginous material formed by these cells being of the hard variety, not staining with thionin, and staining yellow with picro-carmine, etc. By the increase of such nests and the assimilation of the intermediate fibrous material, the original fibro-cartilage was converted into the close-set semi-hyaline cartilage of the trabeculæ and auditory capsules, in which the fibrous material still marks out by its staining-reaction the limits of the cell-clusters.
Such I gather to be Schaffer's conclusions, and they are certainly borne out by my own and Miss Alcock's observations. As far as we have had an opportunity of observing at present, the first process at transformation appears to consist of the invasion of the fibrous tissue of the cranial wall by groups of cells which form nests of cells between the fibrous strands. These nests of cells form round themselves capsular material, and thus form cell-territories of cartilage, which squeeze out and assimilate the surrounding fibrous tissue, until at last all that remains of the original fibrous matrix is the lines of cement-substance which mark out the limits of the various cell-groups.
At present I am inclined to think that both soft and hard cartilage originate in a very similar manner, viz. by the formation of capsular material around the invading chondroblasts, and that the difference in the resulting cartilage is mainly due to the difference in chemical composition of the matrix of the connective tissue which is invaded. Thus the difference may be formulated as follows:—
The hard cartilage is formed by the invasion of chondroblasts into a fibrous tissue, which contains a gelatinous rather than a mucoid substratum, in contradistinction to the soft cartilage which is formed, probably also by the invasion of chondroblasts, in a tissue—the muco-cartilage—which contains a specially mucoid substratum.
Such, then, is the very clearly defined starting-point of the vertebrate skeleton—two distinct formations of different histological and chemical structure,—the one forming a segmented branchial skeleton, the other a non-segmented basi-cranial skeleton.
The Cartilaginous Skeleton of Limulus.
Among the whole of the invertebrates at present living on the earth, is there any sign of an internal cartilaginous skeleton that will give a direct clue to the origin of the primitive vertebrate skeleton? The answer to this question is most significant: only one animal among all those at present known possesses a cartilaginous skeleton, which is directly comparable with that of Ammocœtes, and here the comparison is very close—only one animal among the thousands of living invertebrate forms, and that animal is the only representative still surviving of the palæostracan group, which was the dominant race when the vertebrate first made its appearance. The Limulus, or king-crab, possesses a segmented branchial internal cartilaginous skeleton (Fig. 53, A), made up of the same kind of cartilage as the branchial skeleton of Ammocœtes, confined to the mesosomatic or branchial region, just as in Ammocœtes, forming, as in Ammocœtes, cartilaginous bars supporting the branchiæ, and these bars are situated externally to the branchiæ, as in Ammocœtes. In addition this animal possesses a basi-cranial internal semi-cartilaginous unsegmented plate known as the entosternite or plastron situated, with respect to the œsophagus, similarly to the position of the trabeculæ with respect to the infundibulum in Ammocœtes. Moreover, the cartilaginous cells in this tissue differ from those in the branchial region, in precisely the same manner as the hard cartilage differs from the soft in Ammocœtes.
This plastron, it is true, is found in other animals, all of which are members of the scorpion tribe, except in one instance, and this, strikingly enough, is the crustacean Apus—a strange primitive form, which is acknowledged to be the nearest representative of the Trilobita still living on the earth. None of these forms, however, possess any sign of an internal cartilaginous branchial skeleton, such as is possessed by Limulus. Scorpions, Apus, Limulus, are all surviving types of the stage of organization which had been reached in the animal world when the vertebrate first appeared.
The Mesosomatic or Respiratory Skeleton of Limulus, composed of Soft Cartilage.
Searching through the literature of the histology of the cartilaginous tissues in invertebrate animals, to see whether any cartilage had been described similar to that seen in the branchial cartilages of Ammocœtes, and whether such cartilage, if found, arose in a fibrous tissue resembling muco-cartilage, I was speedily rewarded by finding, in Ray Lankester's article on the tropho-skeletal tissues of Limulus, a picture of the cartilage of Limulus, which would have passed muster for a drawing of the branchial cartilage of Ammocœtes. This clue I followed out in the manner described in my former paper in the Journal of Anatomy and Physiology, and mapped out the topography of this remarkable tissue.
Limulus, like other water-dwelling arthropods, breathes by means of gills attached to its appendages. These gill-bearing appendages are confined to the mesosomatic region, as is seen in Fig. 59; and these appendages are very different to the ordinary locomotor appendages, which are confined to the prosomatic region. Each appendage, as is seen in Fig. 58, consists mainly of a broad, basal part, which carries the gill-book on its under surface; the distal parts of the appendage have dwindled to mere rudiments and still exist, not for locomotor purposes, but because they carry on each segment organs of special importance to the animal (see Chapter XI.). As is seen in Fig. 58, the basal parts of each pair of appendages form a broad, flattened paddle, by means of which the animal is able to swim in a clumsy fashion. Very striking and suggestive is the difference between these gill-bearing mesosomatic appendages and the non-gill-bearing locomotor appendages of the prosoma.
At the base of each of these appendages, where it is attached to the body of the animal, the external chitinous surface is characterized by a peculiar stumpy, rod-like marking, and upon removing the chitinous covering, this surface-appearance is seen to correspond to a well-marked rod of cartilage (Br.C.), which extends from the body of the animal well into each appendage. This bar of cartilage arises on each side from the corresponding entapophysis (Ent.), which is the name given to a chitinous spur which projects a short distance (Fig. 58, B) into the animal from the dorsal side, for the purpose of giving attachment to various segmental muscles. These entapophyses are formed by an invagination of the chitinous surface on the dorsal side and are confined to the mesosomatic region, so that the mesosomatic carapace indicates, by the number of entapophyses, the number of segments in that region, in contradistinction to the prosomatic carapace, which gives no indication on its surface of the number of its components.
Each entapophysis is hollow and its walls are composed of chitin; but from the apex of each spur there stretches from spur to spur a band of tissue, called by Lankester the entapophysial ligament (Ent.l.) (Fig. 58), and in this tissue cartilage is formed. Isolated cartilaginous cells, or rather groups of cells, are found here and there, but a concentration of such groups always takes place at each entapophysis, forming here a solid mass of cartilage, from which the massive cartilaginous bar of each branchial appendage arises.
Further, not only is this cartilage exactly similar to parenchymatous cartilage, as it occurs in the branchial cartilages of Ammocœtes, but also its matrix stains a brilliant purple with thionin in striking contrast to the exceedingly slight light-blue colour of the surrounding perichondrium. In its chemical composition it shows, as might be expected, that it is a cartilage containing a very large amount of some mucin-body.
The Muco-cartilage of Limulus.
The resemblance between this structure and that of the branchial bars of Ammocœtes does not end even here, for, as already mentioned, the cartilage originates in a peculiar connective tissue band, the entapophysial ligament, and this tissue bears the same relation in its chemical reactions to the ordinary connective tissue of Limulus, as muco-cartilage does to the white fibrous tissue of Ammocœtes. The white connective tissue of Limulus, as already stated, resembles that of the vertebrate more than does the connective tissue of any other invertebrate, and, similarly to that of Ammocœtes, does not stain, or gives only a light-blue tinge with thionin. The tissue of the entapophysial ligament, on the contrary, just like muco-cartilage, takes on an intense purple colour when stained with thionin. It possesses a mucoid substratum, just as does muco-cartilage, and in both cases a perfectly similar soft cartilage is born from it.
Fig. 59.—Diagram of Limulus, to show the Nerves to the Appendages (1-13) and the Branchial Cartilages.
One difference, however, exists between the branchial cartilages of these two animals; the innermost axial layer of the branchial bar of Limulus is very apt to contain a specially hard substance, apparently chalky in nature, so that it breaks up in sections, and gives the appearance of a broken-down spongy mass; if, however, the tissue is first placed in a solution of hydrochloric acid, it then cuts easily, and the whole tissue is seen to be of the same structure throughout, the main difference being that the capsular spaces in the axial region are much larger and much more free from cell-protoplasm than are those of the smaller younger cells near the periphery.
I have attempted in Fig. 53 to represent this close resemblance between the segmented branchial skeleton of Limulus and of Ammocœtes, a resemblance so close as to reach even to minute details, such as the thinning out of the cartilage in the subchordal bands and entapophysial ligaments respectively between the places where the branchial bars come off.
Fig. 60.—Diagram of Ammocœtes cut open to show the Lateral System of Cranial Nerves V., VII., IX., X., and the Branchial Cartilages.
In Fig. 59 I have shown the prosoma and mesosoma of Limulus, and indicated the nerves to the appendages together with the mesosomatic cartilaginous skeleton.
In Fig. 60 I have drawn a corresponding picture of the prosomatic and mesosomatic region of Ammocœtes with the corresponding nerves and cartilages. In this figure the animal is supposed to be slit open along the ventral mid-line and the central nervous system exposed.
The Prosomatic Skeleton of Limulus, composed of Hard Cartilage.
The rest of the primitive vertebrate skeleton arose in the prosomatic region, and formed a support for the base of the brain. This skeleton was composed of hard cartilage, and arose in white fibrous tissue containing gelatin rather than mucin.
Is there, then, any peculiar tissue of a cartilaginous nature in Limulus and its allies, situated in the prosomatic region, which is entirely separate from the branchial cartilaginous skeleton, which acts as a supporting internal framework, and contains a gelatinous rather than a mucoid substratum?
It is a striking fact, common to the whole of the group of animals to which our inquiries, deduced from the consideration of the structure of Ammocœtes, have, in every case, led us in our search for the vertebrate ancestor, that they do possess a remarkable internal semi-cartilaginous skeleton in the prosomatic region, called the entosternite or plastron, which gives support to a large number of the muscles of that region; which is entirely independent of the branchial skeleton, and differs markedly in its chemical reactions from that cartilage, in that it contains a gelatinous rather than a mucoid substratum.
In Limulus it is a large, tough, median plate, fibrous in character, in which are situated rows and nests of cartilage-cells. The same structure is seen in the plastron of Hypoctonus, of Thelyphonus, and to a certainty in all the members of the scorpion group. Very different is the behaviour of this tissue to staining from that of the branchial region. No part of the plastron stains purple with thionin; it hardly stains at all, or gives only a very slight blue colour. In its chemical composition there is a marked preponderance of gelatin with only a slight amount of a mucin-body. In some cases, as in Hypoctonus (Fig. 57, B) and Mygale, the capsules of the cartilage-cells stain a deep yellow with hæmatoxylin and picric acid, while the fibres between the cell-nests stain a blue-brown colour, partly from the hæmatoxylin, partly from the picric acid.
All the evidence points to the plastron as resembling the basi-cranial skeleton of Ammocœtes in its composition and in the origin of its cells in a white fibrous tissue. What, then, is its topographical position? It is in all cases a median structure lying between the cephalic stomach and the infra-œsophageal portion of the central nervous system, and in all cases it possesses two anterior horns which pass around the œsophagus and the nerve-masses which immediately enclose the œsophagus (Fig. 61, A). These lateral horns, then, which lie laterally and slightly ventral to the central nervous system, and are called by Ray Lankester and Benham the sub-neural portion of the entosternite, are very nearly in exactly the position of the racquet-shaped head of the trabeculæ in Ammocœtes. It is easy to see that, with a more extensive growth of the nervous material dorsally, such lateral horns might be caused to take up a still more ventral position. Now, these two lateral horns of the plastron of Limulus are continued along its whole length so as to form two thickened lateral ridges, which are conspicuous on the flat surface of the rest of this median plate. In other cases, as in the Thelyphonidæ, the plastron consists mainly of these two lateral ridges or trabeculæ, as they might be called, and Schimkéwitsch, who more than any one else has made a comparative study of the entosternite, describes it as composed in these animals of two lateral trabeculæ crossed by three transverse trabeculæ. I myself can confirm his description, and give in Fig. 61, B, the appearance of the entosternite of Thelyphonus or of Hypoctonus. The supra-œsophageal ganglia and part of the infra-œsophageal ganglia fill up the space Ph.; stretching over the rest of the infra-œsophageal mass is a transverse trabecula, which is very thin; then comes a space in which is seen the rest of the infra-œsophageal mass, and then the posterior part of the plastron, ventrally to which lies the commencement of the ventral nerve-cord.
Fig. 61.—A, Entosternite of Limulus; B, Entosternite of Thelyphonus.
Ph., position of pharynx.
In these forms, in which the central nervous system is more concentrated towards the cephalic end than in Limulus, the whole of the concentrated brain-mass is separated from the gut only by this thin transverse band of tissue. Judging, then, from the entosternite of Thelyphonus, it is not difficult to suppose that a continuation of the same growth of the brain-region of the central nervous system would cause the entosternite to be separated into two lateral trabeculæ, which would then take up the ventro-lateral position of the two trabeculæ of Ammocœtes.
On the other hand, it might be that two lateral trabeculæ, similar to those of Thelyphonus and situated on each side of the central nervous system, were the original form from which, by the addition of transverse fibres running between the gut and nervous system, the entosternite of Thelyphonus and of the scorpions, etc., was formed. From an extensive consideration of the entosternite in different animals, Schimkéwitsch has come to the conclusion that this latter explanation is the true one. He points out that the lateral trabeculæ can be distinguished from the transverse by their structure, being much more cellular and less fibrous, and the cell-cavities more rounded, or, as I should express it, the two lateral trabeculæ are more cartilaginous, while the transverse are more fibrous. Schimkéwitsch, from observations of structure and from embryological investigations, comes to the conclusion that the entosternite was originally composed of two parts—
1. A transverse muscle corresponding to the adductor muscle of the shell of certain crustaceans, such as Nebalia.
2. A pair of longitudinal mesodermic tendons, which may have been formed originally out of a number of segmentally arranged mesodermic tendons, and are crossed by the fibrils of the transverse muscular bundles.
These paired tendons of the entosternite he considers to correspond to the intermuscular tendons, situated lengthways, which are found in the ventral longitudinal muscles of most arthropods.
It is clear from these observations of Schimkéwitsch, that the essential part of the entosternite consists of two lateral trabeculæ, which were originally tendinous in nature and have become of the nature of cartilaginous tissue by the increase of cellular elements in the matrix of the tissue: these two trabeculæ function as supports for the attachment of muscles, which are specially attached at certain places. At these places transverse fibres belonging to some of the muscular attachments cross between the two longitudinal trabeculæ, and so form the transverse trabeculæ.
I entirely agree with Schimkéwitsch that the nests of cartilage-cells are much more extensive in, and indeed nearly entirely confined to, these two lateral trabeculæ in the entosternite of Hypoctonus. Ray Lankester describes in the entosternite of Mygale peculiar cell-nests strongly resembling those of Hypoctonus, and he also states that they are confined to the lateral portions of the entosternite.
From this evidence it is easy to see that that portion of the basi-cranial skeleton known as the trabeculæ may have originated from the formation of cartilage in the plastron or entosternite of a palæostracan animal. Such an hypothesis immediately suggests valuable clues as to the origin of the cranium and of the rest of the basi-cranial skeleton—the parachordals and the auditory capsules. The former would naturally be a dorsal extension of the more membranous portion of the plastron, in which, equally naturally, cartilaginous tissue would subsequently develop; and the reason why it is impossible to reduce the cranium into a series of segments would be self-evident, for even though, as Schimkéwitsch thinks, the plastron may have been originally segmented, it has long lost all sign of segmentation. The latter would be derived from a second entosternite of the same nature as the plastron, but especially connected with the auditory apparatus of the invertebrate ancestor. The following out of these two clues will be the subject of a future chapter.
In our search, then, for a clue to the origin of the skeletal tissues of the vertebrate we see again that we are led directly to the palæostracan stock on the invertebrate side and to the Cyclostomata on that of the vertebrate; for in Limulus, the only living representative of the Palæostraca, and in Limulus alone, we find a skeleton marvellously similar to the earliest vertebrate skeleton—that found in Ammocœtes. Later on I shall give reasons for the belief that the earliest fishes so far found, the Cephalaspidæ, etc., were built up on the same plan as Ammocœtes, so that, in my opinion, in Limulus and in Ammocœtes we actually possess living examples allied to the ancient fauna of the Silurian times.
Summary.
The skeleton considered in this chapter is not the notochord, but that composed of cartilage. The tracing downwards of the vertebrate bony and cartilaginous skeleton to its earliest beginnings leads straight to the skeleton of the larval lamprey (Ammocœtes), in which vertebræ are not yet formed, but the cranial and branchial skeleton is well marked.
The embryological and phylogenetic histories are in complete unison to show that the cranial skeleton is older than the spinal, and this primitive branchial skeleton is also in harmony with the laws of evolution, in that its structure, even in the adult lamprey (Petromyzon), never gets beyond the stage characteristic of embryonic cartilage in the higher vertebrates.
The simplest and most primitive skeleton is that found in Ammocœtes and consists of two parts: (1) a prosomatic, (2) a mesosomatic skeleton.
The prosomatic skeleton forms a non-segmented basi-cranial skeleton of the simplest kind—the trabeculæ and the parachordals with their attached auditory capsules, just as the embryology of the higher vertebrates teaches us must be the case. There in the free-living, still-existent Ammocœtes we find the manifest natural outcome of the embryological history in the shape of simple trabeculæ and parachordals, from which the whole complicated basi-cranial skeleton of the higher vertebrates arose.
The mesosomatic skeleton, which is formed before the prosomatic, consisted, in the first instance, of simple branchial bars segmentally arranged, which were connected together by a longitudinal subchordal bar, situated laterally on each side of the notochord. These simple branchial bars later on form the branchial basket-work, which forms an open-work cage within which the branchiæ are situated.
The cartilages which compose these two skeletons respectively are markedly different in chemical constitution, in that the first (hard cartilage) is mainly composed of chondro-gelatin, the second (soft cartilage) of chondro-mucoid material.
The same kind of difference is seen in the two kinds of connective tissue which are the forerunners of these two kinds of cartilage. Thus, the cranial walls in Ammocœtes are formed of white fibrous tissue, an essentially gelatin-containing tissue; at transformation these are invaded by chondro-blasts and the cartilaginous cranium, formed of hard cartilage, results. On the other hand, the forerunner of the branchial soft cartilage is a very striking and peculiar kind of connective tissue loaded with mucoid material, to which the name muco-cartilage has been given.
The enormous interest of this muco-cartilage consists in the fact that it forms very well-defined plates of tissue, entirely confined to the head-region, which are not found in any higher vertebrate, not even in the adult form Petromyzon, for every scrap of the tissue as such disappears at transformation.
It is this evidence of primitive non-vertebrate tissues, which occur in the larval but not in the adult form, which makes Ammocœtes so valuable for the investigation of the origin of vertebrates.
The evidence, then, is extraordinarily clear as to the beginnings of the vertebrate skeletal tissues.
In the invertebrate kingdom true cartilage occurs but scantily. There is a cartilaginous covering of the brain of cephalopods. It is never found in crabs, lobsters, bees, wasps, centipedes, butterflies, flies, or any of the great group of Arthropoda, except, to a slight extent, in some members of the scorpion group, and more fully in one single animal, the King-crab or Limulus: a fact significant of itself, but still more so when the nature of the cartilage and its position in the animal is taken into consideration, for the identity both in structure and position of this internal cartilaginous skeleton with that of Ammocœtes is extraordinarily great.
Here, in Limulus, just as in Ammocœtes, an internal cartilaginous skeleton is found, composed of two distinct parts: (1) prosomatic, (2) mesosomatic. As in Ammocœtes, the latter consists of simple branchial bars, segmentally arranged, which are connected together on each side by a longitudinal ligament containing cartilage—the entapophysial ligament. This cartilage is identical in structure and in chemical composition with the soft cartilage of Ammocœtes, and, as in the latter case, arises in a markedly mucoid connective tissue. The former, as in Ammocœtes, consists of a non-segmental skeleton, the plastron, composed of a white fibrous connective tissue matrix, an essentially gelatin-containing tissue, in which are found nests of cartilage cells of the hard cartilage variety.
This remarkable discovery of the branchial cartilaginous bars of Limulus, together with that of the internal prosomatic plastron, causes the original difficulty of deriving an animal such as the vertebrate from an animal resembling an arthropod to vanish into thin air, for it shows that in the past ages when the vertebrates first appeared on the earth, the dominant arthropod race at that time, the members of which resembled Limulus, had solved the question; for, in addition to their external chitinous covering, they had manufactured an internal cartilaginous skeleton. Not only so, but that skeleton had arrived, both in structure and position, exactly at the stage at which the vertebrate skeleton starts.
What the precise steps are by which chitin-formation gives place to chondrin-formation are not yet fully known, but Schmiedeberg has shown that a substance, glycosamine, is derivable from both these skeletal tissues, and he concludes his observations in the following words: "Thus, by means of glycosamine, the bridge is formed which connects together the chitin of the lower animals with the cartilage of the more highly organized creations."
The evidence of the origin of the cartilaginous skeleton of the vertebrate points directly to the origin of the vertebrate from the Palæostraca, and is of so strong a character that, taken alone, it may almost be considered as proof of such origin.