Parasynaptic stages in the testis of Aneides lugubris (Hallowell)

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Vol. 11, No. 15, pp. 51 1-528, pis. 25-26, 1 text fig. April 15, 1314

BY

HARRY JAMES SNOOK AND J. A. LONG

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Vol. 11, No. 15, pp. 511-528, pis. 25-26, 1 text fig. April 15, 1914

PARASYNAPTIC STAGES IN THE TESTIS
OF ANEIDES LUGUBRIS (HALLOWELL)

BY

HAEEY JAMES SNOOK AND J. A. LONG

INTRODUCTION

In many of the theories proposed to account for the segre-
gation of hereditary factors a more or less specific association
between the factors and the chromatic elements of the cell is
assumed. Any judgment as to the tenability of such theories
ought to be based, at least in part, upon the behavior of the
chromosomes themselves, the phenomena of synapsis being par-
ticularly significant in this connection. The present study has
been undertaken in an effort to obtain as much evidence as
possible about the actual conditions during the synaptic period.
Inquiries into the spermatogenesis of amphibians have contrib-
uted much towards a solution of the problem, and it has seemed
worth while to extend the observations to another representative
of the same class. An attempt has been made to answer the
following questions : Is there a stage, or a series of stages, in
spermatogenesis during which two or more chromosomes unite,
or in any manner become very closely associated ? If so, in what
manner and to what extent does the process take place, and what
is the subsequent fate of the elements which have been joined
together? This inquiry is limited to the synaptic period and
the stages which precede or immediately follow it.

The urodele, Aneides (=Autodax) lugubris (Hallowell), was
chosen as the subject of this investigation for the following

•« % : :» •- .•:•>• ;;,,.;> • * *••

512 University of California Publications in Zoology [VOL. 11

reasons: (1) The male sex cells are large; (2) the animal is
fairly common in this vicinity and may be obtained throughout
the entire year; and (3) the seriation of stages within the testes
is easily followed because all phases are frequently found in a
single testis, beginning at the anterior end with lobules of sper-
matogonia and passing posteriorly to a region occupied by mature
spermatozoa. Moreover, the cells in any particular lobule appear
to have reached approximately the same stage in differentiation.

The material used for this research was obtained within a
radius of five miles from Berkeley, with the exception of a few
specimens from Twin Peaks, San Francisco, and much of it was
secured upon the campus of the University of California. The
salamanders may readily be found under stones and logs or in
crevices of the rocks. They have also been taken in burrows
at least a foot below the surface of the soil, but in all such cases
observed the burrow opened beneath a rock. It is difficult to
find them during the dry season, as they seek the deeper, moist
levels. According to the investigations of Hitter and Miller
(1899), they breed in June and July. Although no specimens
were collected during June, July, and September, material was
obtained during every other month.

A number of fixing fluids and staining processes were tried
with varying success. Bouin's, Zenker's, and Flemming's fluids
proved most valuable and were extensively employed. Gilson's
fluid seemed to damage the cells of the outer cysts; otherwise,
some very good late prophases were obtained by its use. The
testes were embedded in paraffin and sectioned longitudinally
in order to show the seriation to the best advantage. The sections,
cut from six to twelve micra in thickness, were mounted on slides
in the usual way.

With Bouin's and Zenker's fluids three stains were tried:
iron-alum-haematoxylin with orange G and acid fuchsin as coun-
terstains; safranin followed by gentian violet; and phospho-
tungstic acid haematoxylin. The last seems to be the best for
general purposes, as it stains the growing spermatocyte beauti-
fully without overstaining the mitotic figures. It differentiates
the structures of the cell very clearly, staining the chromosomes
deep blue and the spindles reddish. Many equatorial plates can

19141 Snook-Long: Parasynapsis in Aneides lugubris 513

be excellently demonstrated with safranin and gentian violet
after Zenker's or Bouin's solutions. Material fixed^jn Flem-
ming's solution was stained either with safranin and gentian
violet after the Gram method, or with iron-alum-haematoxylin
and orange G. Gilson's fluid was followed by safranin and
lightgreen.

A few testes, fixed in Flemming 's solution and passed through
the alcohols to seventy per cent, were divided into four or five
parts which were transferred with a drop of alcohol to slides
prepared with egg albumen and then thoroughly crushed and
ground up under a cover glass. The seventy per cent alcohol
was then drawn off and the albumen coagulated by dropping
ninety per cent alcohol upon it, fixing the fragments and loose
cells to the glass. The material thus prepared was carried
through the alcohols and stained either in iron-alum-haema-
toxylin without counterstains, or in safranin with gentian violet.
While many of the cells were broken up, or flattened out, a
considerable number remained intact. Because of their large
size they are somewhat opaque, but most of them are clear enough
for study. This method destroys every evidence of seriation and
for that reason is not of great value by itself, yet, taken in
connection with the sectioned material, it has been found very
useful. This type of preparation, in which the structures are
entire, was most largely used in the determination of the number
of chromosomes previous to the first maturation division.

THE SPERMATOGONIA

The anterior portion of the testis is occupied by spermatogonia
and occasionally lobules may be seen which are filled entirely,
or in part, with cells in the process of division. Among these
it is possible to pick out polar views of the equatorial plate. By
first carefully drawing them with the aid of a camera lucida,
and then counting the chromosomes shown in the resulting sketch,
the number and form were determined. It was not always pos-
sible to demonstrate each element clearly, owing to the sinuous
form of many of the chromosomes and the tendency for them
to gather in clumps, more or less masking those below; and in

514 University of California Publications in Zoology [VOL. 11

many cases it could only be discerned that there were more than
twenty-five and less than thirty. Discarding the latter as doubt-
ful, there still remained nine clear cases in which the number
was definitely twenty-eight, and one case, just as clear, in which
it was only twenty-three.

Plate 25, figure 1, represents this latter and plate 25, figure 2,
illustrates the more usual condition. They are drawn from cells
in one section, located near each other in the same lobule. The
two small elements in the center of the plate in both figures are
quite characteristic of this stage, and, although they cannot
always be found occupying the same relative position, and may
sometimes be widely separated, they can usually be identified
with chromosomes of the same general size and shape lying within
the ring in other spindles. The chromosomes appear to be band-
shaped and are usually bent into the form of a letter U with
the free ends pointing outward. There is considerable variation
in size and shape and in their position with reference to the
spindle.

A split can frequently be discerned in the chromosomes of
the equatorial plate, as is shown by several elements in the cells
figured. It is believed that this is evidence that the plate is
fully formed and that division is about to occur. Many dividing
cells are situated in the immediate vicinity of these two.

The significance of the lesser number shown in figure 1 is
not known, nor is it possible to say just how widely such varia-
tions occur. In the case under consideration there is no evidence
that the condition is in any sense abnormal, for it does not differ
from the others except in the number of chromosomes. This
plate lies in a section twelve micra thick, and the following
section shows only a few tips in the remainder of the cell.

Although such exceptional cases certainly exist, even a casual
exploration of the lobules of dividing spermatogonia will con-
vince one that in a large majority of cases more chromosomes are
present than are delineated in figure 1. In view of the fact that
in every clear case found, with the exception noted, twenty-eight
definite elements could be made out, it seems justifiable to say
that the number of chromosomes in the equatorial plate of
Aneides is usually, but not universally, twenty-eight.

1914] Snook-Long : Parasynapsis in Aneides lugubris 515

THE SPERMATOCYTE

Following the last spermatogonial telophase, the chromatin
becomes very diffuse and is irregularly distributed in the form
of clumps or "net-knots" connected by fine threads (pi. 25,
fig. 3). If there is any arrangement or order in these threads,
it is completely masked by the patches of chromatin, nor is there
any suggestion of orderliness in the size or distribution of the
latter. There is no clear evidence of a " chromoplast " as de-
scribed by Eisen (1900) and later by Janssens (1905) in the
male sex cells of Batrachoseps attenuatu».

Janssens also found evidence of a rotation of the nucleus
during this period, whereby the "chromoplast," at first in the
neighborhood of the sphere, came to lie opposite it. In Aneides
the sphere is found at one side of the nucleus, but further than
that it has been impossible to demonstrate any relation between
it and the elements of the nucleus until the resting stage is past.

In lobules more posterior than those containing the resting
nuclei the fine threads gradually become more pronounced and
can be followed without difficulty for considerable distances. At
the same time it is plain that the threads are of greater diam-
eter and stain more heavily in the region of the sphere than
in the rest of the nucleus. The net-knots appear smaller and
are comparatively few, especially in the immediate vicinity of
the sphere, which may be said to lie at the proximal pole of the
nucleus. In this region the threads appear somewhat con-
densed (pi. 25, fig. 4) and close observation shows that they are
associated two by two in such a manner that when viewed from
the side they resemble the letter V, with the angle pointing
toward the sphere and the diverging arms prolonged in the
opposite direction. They are unbranched, as was also found to
be true for Salamandra by the Schreiners (1906) and for Batra-
choseps by Janssens (1905), the description of the conditions in
Batrachoseps being confirmed by Wilson (1912), who examined
Janssens' preparations. Sections fixed in Bouin's, Zenker's, or
Flemming's solutions and stained in a variety of ways show this
peculiarity. At this stage the distal portion of the nucleus shows
no such polar orientation, and if the sections should be so cut

516 University of California Publications in Zoology [VOL. 11

as to include this part only it could be distinguished with
extreme difficulty, if at all, from the preceding stage.

Comparatively few of the cells examined exhibited the V-
figures when observed from the side, the greater number dis-
playing a Y-shaped arrangement of the threads in the vicinity
of the proximal pole, with the stem of the Y ending in the
immediate neighborhood of the sphere and the arms drawn out
into fine threads which are lost in the distal portion of the nucleus
(pi. 25, fig. 5). In the part of a section which contains sperma-
tocytes in this condition there is a gradual change from the V-
to the Y-figure; the cells characterized by the short-stemmed
Y-figures give place gradually to those in which a long-stemmed
figure predominates (pi. 25, figs. 6 and 7) ; and in the individual
nuclei there is considerable variation in the length of the struc-
ture from the free end to the fork. At times the thick threads
seem also to be double, but it is not possible to demonstrate this
condition in most cases. As the length of the stems of the Ys
increases the extent of the network diminishes.

As can be easily imagined, it is practically impossible to
determine with accuracy in this stage the number of the V- and
Y-figures, and of the fine threads of which they are composed.
While the threads are perfectly clear in side views, they are
easily confused when seen from one end. Nevertheless, in one
nucleus corresponding to figure 4, so cut that the part containing
the pairing threads was viewed from the pole, a diagram indi-
cated the presence of twenty-six to thirty Vs and Ys, or, in other
words, of fifty-two to sixty threads; and in another case in a
stage like that shown in figure 6 there were clearly twenty-eight
thick threads (stems of Ys).

This stage corresponds closely to that figured by Janssens
(1905) in his paper on the spermatogenesis of Batrachoseps and
designated by him the amphitene, in distinction to the preceding
or leptotene condition in which the fine threads are not united.
Much stress is placed upon it by the Schreiners (1906) in their
discussion of the spermatogenesis of Salamandra maculosa as
the stage in which a conjugation of the chromosomes takes place.
Many writers have overlooked it entirely, because, no doubt, of
its close resemblance to the leptotene period and the small number

1914] Snook-Long: Parasynapsis in Aneides lugubris 517

of nuclei usually found in this condition. If the number of
amphitene cells can be taken as an indication of the tturation of
the period, it must be considered as relatively short, because the
cells occupy a much smaller proportion of the testis than either
the spermatogonia or the cells that have advanced to the next
stage.

Posterior to the region in the testis last described are nuclei
containing horseshoe-shaped loops, with their free ends turned
towards the sphere and their bends in the distal half. They appear
somewhat granular (pi. 25, fig. 8 and pi. 26, fig. 10). These loops
have been described in a number of urodeles and would appear
to be very characteristic of spermatogenesis in salamanders. In
thickness they exceed somewhat the thick threads formed by the
union of two fine diverging ones. No trace of the latter can now
be seen. Cross-sections through the proximal portion of the
nucleus show clearly twenty-eight cut ends, demonstrating the
number of loops to be fourteen (pi. 26, fig. 11). Polar views
show the presence of twenty-eight converging threads with their
free ends crowded together in the immediate vicinity of the
sphere and very near to the nuclear membrane (pi. 26, fig. 9).

It will be remembered that this polar orientation is also a
characteristic of the preceding stage, a lateral view showing the
same arrangement of the thick threads forming the stem of the
Y-figure as is here presented by the ends of the loops. This
resemblance is very marked, indeed, and together with the evi-
dence afforded by seriation leads to the conclusion that the loop
is derived from two fine threads wrhich have now completely
disappeared as separate threads and have fused into one.

Up to this point the cells have been gradually increasing in
volume, a fact which makes seriation more certain; but during
the stage of well-developed loops relatively little growth takes
place. As the cells in this condition usually occupy a rather
large portion of the testis, it seems probable that this period lasts
for a much longer time than the amphitene stage.

Later all evidences of polarization are lost and the chromo-
somes end without apparent relation to the position of the centro-
somes or to each other. At the same time a longitudinal split
develops, dividing each loop more or less completely into two

518 University of California Publications in Zoology tv°L- n

portions throughout the greater part of its length, but leaving
one or both of the ends intact (pi. 26, fig. 12), a process which
is the reverse of the one previously described. Cells in which
the split is developing can readily be distinguished from those
in the amphitene stage, by the absence of polarization, by their
larger size, and by their position in the testes near the region -in
which the maturation divisions take place. They are separated
from the amphitene cells by the extensive and clearly defined
region of the polarized loops.

This and the subsequent stages in amphibians have been
described so frequently since first pointed out by Fleming (1887)
that they need not be discussed in detail here. According to
Hermann (1889), Meves (1897), Eisen (1902), Janssens (1901,
1905), Kingsbury (1902), and other careful investigators, the
longitudinal halves of the loop are separated in the first matura-
tion division. This seems to be true in the case of Aneidcs. The
split loops shorten, thicken, and at the same time become twisted
to form what Janssens designates the streptotene stage (pi. 26,
fig. 13). They finally form heterotypical chromosomes, the halves
of which, corresponding to the halves of the split loops, pass to
opposite poles during the following anaphase.

A number of spindles representing the first maturation div-
ision were examined and some drawn with the aid of the camera
lucida (pi. 26, fig. 14). The number of chromosomes was four-
teen, though fifteen were counted in two cases. In the latter,
however, from the position of the chromosomes it seems probable
that a chromosome had just divided and it is possible that four-
teen was the original number in all cases examined. Both sections
and entire cells were employed in making these counts, the latter
being found particularly valuable. While they are frequently
somewhat dense and often pressed out of shape, it is hardly
possible that any of the chromosomes could be lost without rup-
turing the cell membrane. In that case the loss would be detected.

DISCUSSION AND CONCLUSIONS

As previously mentioned, the usual number of chromosomes
found in the equatorial plate of the spermatogonial spindle is

1914] Snook-Long: Parasynapsis in Aneides lugubris 519

twenty-eight. In the stage of the polarized loops, cross-sections
and polar views in the neighborhood of the sphere jiidicate the
number of loops to be fourteen, which agrees with the number of
chromosomes comprising the equatorial plate of the first matura-
tion division. This difference between twenty-eight and four-
teen is to be expected if the chromosomes conjugate whether by
the parasynaptic or telosynaptic method.

Meves (1911) avoids the problem of synapsis by stating : "Die
Geschlechtszellen bezw. ihre Kerne haben nach meiner Vorstel-
lung (1907) die besondere Eigenschaft ererbt, beim Eintritt in
die Wachstumsperiode nur die halbe Zahl von Chromosomen
auszubilden. " To which Wilson (1912) replies, "Certainly the
adoption of this simple solution would save a great deal of
trouble; but I fear that the facts compel us to take a more
roundabout way out of our difficulties." A condition so well-
defined and clear as the amphitene stage would seem to be too
significant to be thus lightly considered.

This has been interpreted by different investigators to repre-
sent either a union of two threads (the Schreiners (1906),
Janssens (1905, 1908), Wilson (1911), or a splitting of one
Meves (1908), Goldschmidt (1908), Fick (1908). In Aneides
the evidence afforded by seriation as recapitulated below seems to
the writers to point toward the former interpretation.

1. Fine, unpaired threads become polarized with respect to
that side of the nucleus near which lies the sphere, the proximal
pole.

2. Coincident with this polarization, threads are found asso-
ciated two by twro at the proximal pole of the nucleus.

3. By the association of any two threads there is formed a
figure, which, when observed from a position at right angles to
an axis passing through the poles of the cell, resembles the letter
V with the angle turned toward the sphere.

4. Seriation showys that the V-shaped figures are succeeded by
Y-like figures, of which the stem, frequently exhibiting a double
condition, lies in the region of the centrosomes, while the arms
diverge widely away from the sphere. Furthermore, it may be
observed that as this stage becomes more advanced, the stem of
the Y increases in length at the expense of the arms.

520 University of California Publications in Zoology [VOL. 11

5. More posterior in the testes the Y-figures are replaced by
loops showing the same polarization, i.e., with the free ends
directed toward the sphere while the bends are found in the
region opposite to the centrosomes.

6. At this stage the fine threads can no longer be detected and
the loops appear as single threads.

This evidence leads to the conclusion that the loops are formed
by the union of the fine threads. A more critical examination
of the early stages raises the question as to what are the relations
of the conjugating leptotene threads to each other. Up to this
point the individual V- or Y-figures described in any one nucleus
have been considered as unrelated to each other. If there are
only fourteen bifid figures in any one nucleus, each must be
considered one end of a loop in the later polarized stage, the
other end of the loop not yet having come into existence. But
since there are about twenty-eight pairs of threads at the begin-
ning of polarization and since the twenty-eight free ends of the
completed loops- directed toward the proximal pole correspond
in position to the stems of the Ys, it leads one to think that the
two ends of each loop are formed simultaneously and before the
middle part comes into existence.

While the foregoing is strong evidence that the stem of each
Y-figure becomes one end of a polarized loop, the way in which
the two ends of each loop become associated remains to be con-
sidered. It will be remembered that each one of the fifty-six
short, fine, leptotene threads which unite in one way or another
to form the fourteen loops is indirectly continuous with the others
through the medium of the network. The further evolution of
these threads might be thought of in one of two ways. In the
first place the fifty-six threads might be imagined as separate,
individual filaments which pair to form the twenty-eight Y-
figures; in the second place, they may be conceived of as not so
many independent parts, but as so definitely related that each
branch of each Y would be one end of a potential thread, the
•other end of which would be represented by one of the branches
of some other Y.

In regard to the first of these possibilities, it might be argued
that the arms of the Y-figures on one side meet and join with

1914] Snook-Long : Parasynapsis in Aneides lugubris 521

similar arms of Y's on the other side, giving rise to the loop by
a process of telosynapsis in addition to parasynapsjs^ jjfig. A).
That there is no evidence for such a conclusion is indicated by
the observation that threads may frequently be followed for long
distances around the nucleus and that free ends have never been
found except in the vicinity of the sphere. When the threads
are definitely formed, there are no morphological indications that
an end-to-end union has taken place.

These considerations indicate at once the validity of the
second alternative, for if the threads exhibit no free ends except
at the proximal pole of the nucleus where they unite to form the
Y-figures, it seems reasonable to conclude that they represent
the ends of twenty-eight threads which are evolved from their
ends towards their middle at the expense of the nuclear netwrork.
In other words, each of the fifty-six threads becomes directly
continuous with one only of its own kind by the time that the
loops are complete. If this conception of their origin and nature
be sound, the way in which they become associated in the loops
might be interpreted in one of two ways: (1) The loops may
be formed by the association of two potential threads by a
process which begins at approximately the same time at both
ends (fig. J3). (2) One leptotene thread might be joined at the
ends to two other threads (fig. C). Both of these interpretations
have one feature in common, that of a side-to-side union. There
seems, however, to be little reason to believe that the second
condition actually exists in the male sex cells of Aneides. If
each leptotene thread were joined at the ends to other threads
at the beginning of polarization (fig. C), as the bifid figures
closed up, most of the threads would be drawn together in one
or more points in the region of the distal pole of the nucleus
(fig. D), which is, in fact, the region of widest separation. The
fact that there is no evidence of such behavior upon the part of
the threads would seem to dispose of this interpretation.

Therefore, it seems probable that the number of pairing
threads is twenty-eight and that, as rapidly as they are evolved,
they unite two by two, side by side, and at both ends, the middle
part of the potential threads being lost in the distal half of the
nucleus.

522 University of California Publications in Zoology [VOL. 11

B

G D

Figs. A to D. — Diagrams to illustrate possible modes of association in
synapsis of the fifty-six fine threads (leptotene) which are formed from
the nuclear network and become polarized with regard to the sphere which
lies at the proximal side of the nucleus. The large outer circle represents
the cell wall; the large inner, the nuclear membrane; the small one between
the two large, the sphere.

Fig. A. — Of the fifty-six threads which are imagined as separate, dis-
tinct filaments eight are represented as forming two loops (amphitene)
by a double process of pairing. They form by parallel union four (of the
twenty-eight) Ys which by end to end junction give rise to the loops.
There is no evidence of such a condition.

Fig. B. — Four complete threads are indicated, the eight ends of which,
corresponding to the eight separate threads of figure A, arise separately,
are at first merged into the nuclear network of the distal part of the
nucleus, and with the disappearance of the network become continuous
as four threads. These before completion and whi-le potentially present
in the network become paired in such a manner that the ends of two
threads unite to form two Ys, which by closing up give rise to a single
loop which is unconnected with the other. Fourteen such loops are formed.

Fig. C. — The complete threads arise as in figure B. A different mode
of synapsis is represented in which the two ends of one complete thread
are not paired with the two ends of only one other thread, but with one
end each of two other threads.

Fig. D. — A later stage of the condition shown in figure C, showing
how with the final closing of the Ys the bends of the loops would be drawn
together, a condition not existing.

1914] Snook-Long: Parasynapsis in Aneides lugubris 523

With these facts and considerations in mind, it is concluded
that the V- and Y-figures do not indicate a splitting, _as main-
tained by some, but that they represent a progressive, parallel
union or conjugation of fine (leptotene) threads; in other words,
a parasynaptic union. This union takes place during the amphi-
tene stage and leads to the formation of the single, thick, polarized
loops. There is no reason for confusing the split which does occur
in the stage following the formation of the loops (and wrhich may
be the reverse of parasynapsis) with the union in the amphitene
stage, since the two stages are easily distinguishable both by their
appearance and position in the testis.

The evidence set forth above does not warrant an assertion
that the conjugating leptotene threads are identical with the
spermatogonial chromosomes. Nevertheless, the fact that the
number of chromosomes (tetrads) in the first maturation spindle
is half that of the chromosomes in the spermatogonial division,
together writh the evidence that the tetrads are formed by the
union of threads evolved from the nuclear network which in
turn is formed from the chromosomes of the last spermatogonial
telophase, suggests very strongly that the spermatogonial chro-
mosomes which went into the nuclear network reappear as the
pairing leptotene threads. This idea is further supported by the
manner in which the two branches of two Ys become continuous
as though they were the ends of threads (chromosomes) poten-
tially existing in the network, though not distinguishable as such.
Or, to reverse the conception, if it is considered that the sperma-
togonial chromosomes retain their continuity throughout the
resting stage of the nucleus, then it is easier to comprehend why
the proper 'Y-figures become associated as the leptotene threads
are evolved, for the chromosomes, though in modified form and
only partly distinguishable as threads, begin to pair as they
might if pairing were delayed until they were completely formed.
Although this way of stating the conception is open to the criti-
cism that it is an argument in a circle, still the conception is
worthy of consideration as having some weight on the positive
side of the question of the individuality of the chromosomes.

It has already been pointed out that the stage of the polarized
loops which follows that of the conjugation of the leptotene

524 University of California Publications in Zoology [VOL. 11

threads has a relatively much longer duration than any other of
the stages of the prophase of the first maturation division. If
it is true that the conjugating threads have the value of paired
maternal and paternal chromosomes and constitute the mechan-
ism for the segregation and recombination of mendelian char-
acters or their factors, it is to be noted that there is ample
opportunity for the reconstitution of the chromosomes and for
any interchange of substances composing the chromosomes which
may be concerned in the transmission of inherited characters.

SUMMARY

1. The usual number of chromosomes found in the sperma-
togonia of Aneides lugubris is twenty-eight.

2. The V-figures at the beginning of polarization number
twenty-six to thirty, and Ys at a slightly later stage twenty-eight.

3. The number of polarized loops and of tetrads formed from
the loops is fourteen.

4. Each tetrad is the result of a parasynaptic union of fine
threads.

Transmitted May 3, 1913.

LITERATURE CITED

ElSEN, G.

1900. The spermatogenesis of Batrachoseps. Journ. Morph., 17, 1-117,

pis. 1-14, 12 figs, in text.
FICK, E.

1908. Zur Konjugation der Chromosomen. Arch. f. Zellforsch., 1,

604-611.
FLEMMING, W.

1887. Neue Beitrage zur Kenntnis der Zelle. Arch. f. mikr. Anat.,
29, 389-463, pis. 23-26.

GOLDSCHMIDT, B.

1906. Eeview of work of the Schreiners. Zool. Centralblatt, 13

611-612.
1908. 1st eine parallele Chromosomen-konjugation bewiesen? Arch.

f. Zellforsch, 1, 620-622.
JANSSENS, F. A.

1901. La spermatogenese chez les tritons. La Cellule, 19, 7-116, 3 pis.
1905. Evolution des auxocytes males du Batrachoseps attenuatus. Ibid.,

22, 377-425, 7 pis.

1914] Snook-Long: Parasynapsis in Aneides lugubris 525

JANSSEXS, F. A. ET DUMEZ, E.

1903. L' element nucleinien pendant les eineses de maturation des
spermatocytes chez Batrachoseps attenuatus ~el ~Pleihodon
cinereus. La Cellule, 20, 419-461, 5 pis.
JANSSENS, F. A. ET WILLEMS.

1908. Spermatogenese dans les batraciens, IV. Le spermatogenese
dans 1' Alytes obstetricus. La Cellule, 25, 151-173, 2 pis.

KINGSBURY, B. F.

1902. The spermatogenesis of Desmognathus fusca. Amer. Journ. Anat.,

1, 99-135, pis. 1-4, 1 fig. in text.

MCGREGOR, J. H.

1899. The spermatogenesis of Amphiuma. Journ Morph., 15 (Supple-
ment), 57-104, pis. 4-5.

MEVES, F.

1896. Ueber die Entwicklung der mannlichen Geschlechtzellen von

Salamandra maculosa. Arch. f. mikr. Anat., 48, 1-83, pis. 1-5.

1908. Es gibt keine parallele Konjugation der Chromosomen. Arch.

f. Zellforsch., 1, 612-619, 1 fig. in text.

1911. Chromosomenlangen bei Salamandra, nebst Bemerkung zur
Individualitatstheorie der Chromosomen. Arch. f. mikr.
Anat., 77, Abt. 2, 273-300, pis. 11-12.
MONTGOMERY, T. H.

1903. The heterotypic maturation mitosis in Amphibia and its gen-

eral significance. Biol. Bull., 4, 259-269, 8 figs, in text.

1904. Some observations and considerations upon the maturation

phenomena of the germ cells. Ibid., 6, 137-158, 30 figs.

1911. The spermatogenesis of an hemipteron, Euchistus. Journ.

Morph., 22, 731-798, 5 pis.
BITTER, W. E., and MILLER, L.

1899. A contribution to the life-history of Autodax lugubris Hallow,
a California salamander. Amer. Nat., 33, 691-704, 7 figs, in
text.
SCHREINER, A. und K. E.

1906 a. Neue Studien iiber die Chromatinreif ung der Geschlechtszellen ;
I. Die Eeifung der mannlichen Geschlechtszellen von Tomop-
teris onisciformis. Arch. Biol., 22, 1-69, pis. 1-3, 2 figs, in
text.

1906 b. II. Eeifung der mannlichen Geschlechtszellen von Salamandra
maculosa (Laur.), Spinax niger (Bonap.) und Myxine glutinosa
(L.). Ibid., 22, 419-492, pis. 23-26, 1 fig. in text.
WILSON, E. B.

1912. Studies on chromosomes, VIII. Observations on the maturation-

phenomena in certain Hemiptera and other forms, with con-
siderations on synapsis and reduction. Journ. Exp. Zool., 13,
345-431, 9 pis.

PLATE '25

All the figures are drawn at the same magnification (x 2350) with a
Spencer 2 mm. objective, a Zeiss No. 12 compensating ocular, and an
Abbe camera lucida. Eeduced magnification on plates, 1700 diameters.

Fig. 1. Spermatogonium in the equatorial plate stage, showing
twenty-three chromosomes. Fixed in Zenker's fluid and stained with iron-
alum haematoxylin and orange G.

Fig. 2. Same stage as above but with the usual number of chromo-
somes (twenty-eight). Zenker's fluid, iron-alum haematoxylin, and orange
G.

Fig. 3. Spermatocyte before the beginning of polarization. Fixed in
Zenker's solution and stained with phosphotungstic acid haematoxylin.

Fig. 4. Spermatocyte at the beginning of polarization showing V-
figures. Zenker's fluid and phosphotungstic acid haematoxylin.

Fig. 5. Spermatocyte with short-stemmed Y-figures. Phosphotungstic
acid after Zenker's fluid.

Figs. 6 and 7 illustrate successive steps in the pairing of the threads.
The section from which Fig. 6 was taken was fixed in Zenker's solution
and stained with phosphotungstic acid haematoxylin. Figure 7 was taken
from material fixed in Bouin's fluid and stained with iron-alum haema-
toxylin.

Fig. 8. A lateral view. Loops completed. Zenker's fluid and phos-
photungstic acid haematoxylin.

[526]

UNIV. CALIF PUBL.ZOOL.VOL.il

[SNOOK - LONG] PLATE 25

HJ S.Del

PLATE 26

Fig. 9, 10, and 11. The loops completed.

Fig. 9. Pole view. The sphere lies above the nucleus. The twenty-
eight free ends in the vicinity of the sphere represent fourteen loops.
Zenker's fluid and phosphotungstic acid haematoxylin.

Fig. 10. Lateral view. Zenker's fluid, iron-alum haematoxylin, and
orange G.

Fig. 11. Section of spermatocyte in the loop stage. The loops are
cut twice. Fixed in Zenker's fluid and stained with iron-alum haematoxylin.

Fig. 12. Complete cell from crushed preparation showing the begin-
ning of the split. Flemming's fluid and iron-alum haematoxylin.

Fig. 13. " Strepsinema. " Only a small portion of the cell included
in the section. Gilson 's fluid, iron-alum haematoxylin, and orange G.

Fig. 14. Prophase to first maturation division. Complete cell from
crushed preparation — somewhat flattened. Flemming's fluid and iron-
alum haematoxylin.

[528]

UNIV. CALIF: PUBL. ZOOL.VOL. n

[SNOOK - LONG] PLATE £6
, V

~

H. J.S.Del.

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

5. On the Skeletal Morphology of Gonyaulax catenata (Levander), by

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vestigations, by Charles Atwood Kofoid. Pp. 349-352.

Nos. 8 and 9 in one cover. November, 1911 _ .40

Index, pp. 353-357.

Vol. 9. 1. The Honied Lizards of California and Nevada of the Genera Phryno-
soma and Anota, by Harold C. Bryant. Pp. 1-84, plates 1-9. Decem-
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2. On a Lymphoid Structure Lying Over the Myelencephalon of Lepisos-

tens, by Asa C. Chandler. Pp. 85-104, plates 10-12. December, 1911. .25

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7. Observations on the Suckling Period in tte Guinea-Pig, by J. Marion

Read. Pp. 341-351. September, 1912 10

8. Haeckel's Sethocephalus eucecryphalus (Radiolaria), a Marine Ciliate,

by Charles Atwood Kofoid. Pp. 353-357. September, 1912 .08

Index, pp. 359-365.

Vol. 10. (Contributions from the Museum of Vertebrate Zoology.)

1. Report on a Collection of Birds and Mammals from Vancouver Island,

by Harry S. Swarth. Pp. 1-124, plates 1-4. February, 1912 1.00

2. A New Cony from the Vicinity of Mount Whitney, by Joseph Grinnell.

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Nos. 3 and 4 in one cover. April, 1912 12

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Nos. 6 and 7 in one cover. May, 1912 15

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Swarth. Pp. 197-406, pis. 6-10. October, 1913 2.00

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2. On the Structure and Relationships of Dinosphaera palustris (Lemm.),

by Charles Atwood Kofoid and Josephine Rigdea Michener. Pp. 21-

28. December, 1912 10

3. A Study of Epithelioma Contagiosum of the Common Fowl, by

Clifford D. Sweet. Pp. 29-51. January, 1913 .25

4. The Control of Pigment Formation in Amphibian Larvae, by Myrtl*

E. Johnson. Pp. 63-88, plate 1. March, 1913 .86

5. Sagitta calif arnica, n. sp., from the San Diego Region, including

Remarks on Its Variation and Distribution, by Ellis L. Michael.

Pp. 89-126, plate 2. June, 1913 .35

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Amphibians, by S. J. Holmes. Pp. 143-154, plates 5-6.

8. Behavior of Ectodermic Epithelium of Tadpoles when Cultivated in

Plasma, by S. J. Holmes. Pp. 155-172, plates 7-8.

Nos. 7 and 8 in one cover. September, 1913 30

9. On Some California^ Schizopoda, by H. J. Hansen. Pp. 173-180, pi. 9.

November, 1913 10

10. Fourth 1'axonomic Report on the Copepoda of the San Diego Region,

by Calvin O. Esterly. Pp. 181-196, pis. 10-12. November, 1913 _. .16

11. The Behavior of Leeches with Especial Reference to Its Modifiability,

A. The General Reactions of the Leeches Dina microstoma Moore and
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the Leech Dina microstoma Moore, by Wilson Gee. Pp. 197-305, 13
text figures. December, 1913 — 1.00

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Little. Pp. 307-328, plates 13-15. February, 1914 20

13. Modifications and Adaptations to Functions in the Feathers of Circus

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14. A Determination of the Economic Status of the Western Meadowlark

(Sturnella neglecta) in California, by Harold Child Bryant. Pp. 377-

510, plates 21-24, 5 text figures. February, 1914 1.25

15. Parasynaptic Stages in the Testis of Aneides lugulris (Hallowell), by

Harry James Snook and J. A. Long. Pp. 511-528, plates 25-26, 1 text

fig. April, 1914 25

Vol. 12. 1. A Study of a Collection of Geese of the Branta canadensis Group from
the San Joaquin Valley, California, by Harry S. Swarth. Pp. 1-24,
plates 1-2, 8 text figs. November, 1913 SO

2. Nocturnal Wanderings of the California Pocket Gopher, by Harold C.

Bryant. Pp. 25-29, 1 text fig. November, 1913 05

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Rogers Atsatt. Pp. 31-50. November, 1913 20

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with Especial Reference to the Distributional Problems Presented,

by Joseph Grinnell. Pp. 51-294, plates 3-13, 9 text figs. March, 1914. 2.40

Vol. 13. 1. The Schizopoda of the San Diego Region, by Calvin O. Esterly. Pp.

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phora of the San Diego Region, by Calvin O. Esterly. Pp. 21-38.
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