Taxonomic studies in the genera Tribulus and Kallstroemia
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Authors Storm, Gertrude Johanna Schaufler, 1939-
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/319431 TAXONOMIC STUDIES IN THE GENERA TR I BULUS AND KALLSTROEMIA
' : : : : x - ' by Gertrude S. Storm
A Thesis Submitted to the Faculty of the
, DEPARTMENT OF BOTANY In Part ial Fulf11Iment of the Requirements ’ For the Degree o f
MASTER OF SCIENCE In the Graduate College
THE UNIVERSITY OF ARIZONA -
1963 STATEMENT BY AUTHOR
This thesis has been submitted In partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable with out special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quo tation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the material is in the interests of scholar ship. In a ll other instances, however, permission must be obtained from the author.
SIGNED; G&*cX'i+*ksL
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
CHARLES T. MASON , (JJR. / DateT Professor of Botany ACKNOWLEDGMENTS
The author wishes to express her sincere apprecia tion to Dr. Charles T. Mason, J r., Dr. Walter S. P h illip s , and Dr. B. M. Harris for their many helpful suggestions during the course of her graduate study. She is especially indebted to Dr. Mason and Dr. Harris for their help in preparation of the manuscript, and to Dr. L. W. Storm for his patience and help. TABLE OF CONTENTS
Page
LIST OF FIGURES AND TABLES , ...... v
ABSTRACT ...... ;. , ...... vi
INTRODUCTION ...... 1
GERMINATION STUDIES...... 5
Experiment I » ...... 6 Experiment II . . , ...... „ ...... 7 Experiment III...... 9 D i s cu ssi on o.e.o.oo.-...... 1 0
CYTOLOGICAL STUDIES...... 12
CHROMATOGRAPHY STUDIES ...... 13
POLLEN VIABILITY STUDIES ...... 26
S UMMARY 6 0 9 0 O O 6 6 0 . 0 O . . 0,0 O O 6 . O . . 0 . 26
LITERATURE CITED ...... 30
i v LIST OF FIGURES AND TABLES
FIgure Page
1. Rf values from a chromatogram of an unknown species of Kal 1 stroemia ...... , ...... 16 - , ■ . ■ ;■ ' ...... 2. Rf values from a chromatogram of TrIbulus te rre s tr Is ...... 17
3. Rf values from a chromatogram of Kal1stroemia grand I flo ra ...... 18
4. Rf values from a chromatogram of K. grand!flora. , ...... 19
5. Rf values from a chromatogram of K. calIfo rn lca...... 20 . . - — — — ------6. Rf values from a chromatogram of K. parvlflora ...... 21
7. Rf values from a chromatogram of K. parvl flo ra » . . , . , ...... 22 , — ■ ■■ . " , , 8. Rf values from a chromatogram of K. hi rsut Iss l.ma ...... 23 •
Table ■ - , . • : : . . ■. ' ,■ ■ ,
1. Results of eight treatments of fruit segments of Kal1stroemia grand!flora ...... 8
2. Per cent viability of pollen of the four ArI zona Kal1stroemi a spec I as...... 26
v ABSTRACT
The relationship between two genera in the
Zygophyl1aceae was explored by means of paper chromatography of leaves and embryos, karyotype studies, and pollen viabil
ity studjes of Tribu1 us terrestris, Ka11stroemia grand!flora,
K. parvi flora, J<„ cali fornica, and K„ hirsutisslma. A num- ber of treatments were used in an attempt to germinate seeds of Kailstroemi a. Radicle extrusion occurred on some fruit segments which had been subject to high temperature and suf ficient moisture, but the radicles quickly became contami nated. A count of the chromosomes in root tips of Tribulus te rres tr is showed a 2n number of 24. Attempts to determine the karyotype of Kalistroemia grand?flora using pollen mother cells were not successful. Chromatograms of leaves and embryos of K. grand!flora, K. parvi flo ra , and K. cal i forn i ca s how a s t r i k i h g si mi' 1 a r it y . Tribul us ter rest ri s
is also very similar to these taxa. Chromatograms of
K. hi rsutissima differed in number and placing of spots. A study of pollen grains from herbarium specimens showed that the four taxa of Kalistroemia in Arizona possess a very high degree of pollen viability. These results indicate a close relationship between the three taxa of Kal1stroemi a and Trlbulus te rre s trfs . They do not support separation of the three taxa of Kalistroemia into species, or of Tribulus and
Kal1stroemia into two genera. INTRODUCTION
Members of two genera of the Zygophyl1aceae now generally accepted as separate, fribulus and Kal1stroemia, were f ir s t described by Linnaeus (11) as belonging to one genus, Tribulus, in the class Decandria, Monoqynia. He recognized four species: T« maximus,, T. 1 anuqinosus,
T. te rr e s tris , and cistoides.
Scopoli (I? ) s p lit Linnaeus' Tribulus into two genera oh a basis of the fruit structure. He described the genus
Tribulus as having a schizocarp breaking up into five parts of two locules each, with each locule containing one or two seeds, and Kal1stroemia as having a schizocarp of ten seg ments, each with one embryo.
This criterion of fruit structure provides the present basis for separation of the two genera (9, 22). Three of
Linnaeus' original Tribulus species are still recognized as val id: T. te rre s tr Is , T, cistoides, and T_. lanuq? nosus.
The fourth is now named J<. maxima (L. ) Torrey and Gray. The author's discussion of the two genera is confined to the one species of Tribulus, £° terrestr i s, and the four species of
Kal1stroemia, K. qrandiflora, K, californica, K. parviflora, and K. h i rsutissima, which occur in Arizona.
:i 2
Tr?bulus te rre s tris is native to the Sahara Desert, and was probably introduced into the United States around i860 by importation of livestock to the range lands of the midwest (8). It has spread rapidly, and is now found from
C alifornia to the Southern A tlan tic seaboird. T. te rre s tris is classified as a noxious weed because of two long sharp spines on each fr u it segment, which have given rise to the common names of “goat headcli and “puncture v in e .” It is a prostrate annual in Arizona, with 8-12 leaflets pinnately arranged, and small, yellow, a x illa ry , s o lita ry , peduncled flowers.
Kal1stroemia grand!f1 ora was first described in 1852 by Torrey, who saw it in Hooker's herbarium and published his description of it with the present specific epithet in
Gray9s nPlantae Wrightianae” (7)> Where its distribution is given as "borders of the Gila,n Its center of dispersal is not known. K. grand if io ta , di ffers from the other species of
Kal1stroemia in Arizona by having larger petals (15-30 mm), long style (beak) (8-11 mm), and persistent sepals,
Watson (23) described K. californica as Iribulus c a lifo rn icu s, having a " fru it deeply lobed, . . . short-beaked, the carpels with four or five stout obtuse tubercles upon the back; . . . collected by Dr. E. Palmer, in Lower C alifornia
(1 S/D)> on the eastern side of the peninsula." Vail (21) published the present cbmbination in 1895, giving the distri bution as “Arizona, Northern Mexico and Lower C alifo rn ia ."
If this is properly a Kal1stroemia, as it has been classified since V a il‘ s combination was published, it should have ten carpels. Watson8s description of the fruit as being "deeply five-lobed" is of interest. Brewer and Watson (4) later refer to T. cali fornicus as being a "true Tribulus" with five carpels. Examination of the original specimen would be. of value. The taxon which at present is called K. cal iforni.ca has elevated and sharp dorsal tubercles on the fr u it segments> small petals (less than 12 mm), beak of less than 3 mm, and sepals deciduous before the fr u it matures.
Kal1stroemia p a rv iflo ra , which had been collected in
Mississippi and Texas, was described by Norton (13) in 1898 as a new species. It has a beak 5~8 mm long, persistent sepals, and petals 6-8 mm long. Norton described it as being
"nearest K. grand!flora of the Southwest U.S., but differs from that species in the smaller leaves with fewer leaflets and smaller flowers,"
Kal1stroemia hi rsutissima differs from K. californica in its abundant pubescence on the leaves and stems. Length of its beak is less than 3 mm, and sepals are persistent. It was first described by A. M. Vail i n i 903 in Small's EBFlora of the Southeastern U .S." Its range at that time was given as Kansas and Colorado, to Texas, New Mexico and Mexico (18). This thesis is an attempt to explore the relationship of the two genera. The only consistent difference between them is the appearance of the fr u it and the number of fr u it segments. Flower size, growth habit, and all other vegeta tive characteristics are not consistently distinct enough to be used as criteria for separation. Some other basis for judgment was sought, and the author chose two approaches.
The f ir s t was to compare the chromosome morphology of
Tribulus terrestris with that of Kal1stroemia grand!flora, the most common spec Ies o f Ka11s t roem i a in Arizona, and the most easily distinguished morphologically from the other species of Kal1stroemia and from Tribulus.
The second approach was to compare chromatograms of leaves and embryos of the four species of KalTstroemia with
Tribulus to determine if a combination of patterns of ami no acids characteristic of Tribulus and one of the species of
Kal1stroemia could be found in one of the other species of
Kal1stroemia, thus indicating a possible combination of genetic material.
A constant supply of fresh material would be of value
in the above studies, so attempts were made to germinate and grow Kal1stroemia seeds in a controlled environment.
Since pollen viability is one means of detecting a possible hybrid (2), and it is relatively easy to check, this was done by using herbarium specimens of the four Kal1stroemia species. GERMINATION STUDIES
The four species of Kal1stroemia found in Arizona have a natural growing period of only three months, August through October. Abil ity to grow them in a control led environment would facilitate collection of buds and root tips
for studies of meiotic and mitotic figures, and would permit extended hybridization attempts. A number of other investi gators have attempted to germinate Kalistroemia, but none have been successful (D„ M. Maddox, personal communication;
S. Shel1 horn, personal communication; Al-Katib (1) )V
Kal1stroemia seedlinqs have been found in nature only after two or three summer rains, which occur when daytime temperatures are near 45° C (10), It would seem feasible that a combination of high temperatures and s u ffic ie n t mois
ture would enable one to achieve germination under controlled conditions. Water soluble chemical inhibitors have been
found in a number of plants, so two tests were designed to determine i f a chemical inhibitor were a p artial cause of , difficulty in the germination of Kal1stroemia under controlled condi t ions.
5 6
Experiment I
Twenty-five fr u it segments were placed in a container in running tap water overnight. Segments were then removed and placed on moist f i l t e r paper in a s te rile petri dish under a 100-watt 1ight bulb approximately 18 inches away which was kept on for approximately 12 hours each day. The segments were kept at room temperature. After two weeks, no germination had occurred.
To further test the hypothesis that a water soluble chemical inhibitor might be causing d iffic u lty in germination, f i f t y f r u it segments of Kal1stroemia grand? flo ra were soaked in a watch glass of d is tille d water for four days. The fr u it segments were removed and twenty-five Tribulus fr u it segments were placed in the water. Without treatment,
Tribulus germinates in a few days when placed on moist filte r paper. After fiv e days, the segments were removed and placed on moist filter paper. There was no appreciable difference in either rate or per cdht of germination of the segments when compared with untreated Tr?bu1 us segments.
Attempts to remove the fr u it coats from Ka 11stroemia seeds were not successful. It was not possible, with the materials available, to crack the tough coat without damaging the embryo, Which is closely adhering to the fruit coat. 7
ExperIment I I
Fruit segments used in the following experiments to
determine effects of temperature on germination, unless
otherwise noted, were those of KalIstroemi a grand i f 1 ora which had been collected the previous year.
In the first treatment, a large number of fruit seg
ments were refrigerated at -1° C for th irty days. They were
then placed in coarse sand on an Eberbach shaker at high
speed for three hours. After th is mechanical a g ita tio n , the
segments were put in an oven which could be easily maintained
for four minutes at 68° C» Ten segments were then put in a
s te rile petri dish onto f i l t e r paper kept moist with an
inorganic stock solution. The stock solution used was as
follows: 1.425 g Ca(N03 ) 2»4H20; 105 mg MgSO^TI-^O;
425 mg KNO^; 305 mg KCL; 100 mg KHgPO^; glass d is tille d water to make 1.0 liter. This was diluted to 1/5 for use.
Plates were kept under a 100-watt lig h t for sixteen hours of
lig h t and eight of dark each day. Temperature readings
taken under a glass of comparable thickness to a petri dish
cover showed that the temperature varied from 40-45° C during
the light period, and from 20-25° C during the dark period.
In treatment 2, seeds were not f ir s t refrigerated.
The remainder of the treatment was identical to 1, and ten
segments were put under lig h t. In treatment 3, refrigeration 8 and scari fication were omitted. Treatment k segments received no prior treatment before ten segments were placed under
light as described above.
Treatments 5, 6, and 7 were heated to 78° C for 10,
15, and 5 minutes respectively. This temperature is four degrees above the highest temperature thought to be reached
in Tucson in the summer at ground level, and was as close to
74° C as the oven used could be constantly maintained.
Treatment 8 consisted of placing ten three-year old fr u it segments under lig h t as described above.
The following table shows the results of the above set of experiments.
TABLE 1. Results of eight treatments of fruit segments of Kal1Stroemia grand? flora
T reatment number 10 days after in itia tio n ,1 3 days
1 One radicle extruded No further results
2 One radicle extruded No further results
3 One radicle extruded No further results
' 4 No results
5 Dried, but 9 of 10 with split coats 6 One fr u it coat s p lit
7 One fr u it coat s p lit
8 No results It was possible to easily remove embryos from the segments of the f ir s t treatment a fte r they had been under
lig h t for ten days,.as the fr u it coats had s p lit* Three embryos were placed on complete nutrient agar. Though the agar and the stock solution used for moisture had been auto- claved, the embryos became contaminated and were discarded.
Eventual 1y, a ll of the above became overgrown with bacterial and fungal contaminants. .
In an attempt to prevent contamination, ten fruit segments of Kal1stroemi a grand? f lora whi ch had been treated
in the manner described in treatment 1 were dipped into an undiluted solution of Arasan, a common seed disinfectant.
They were then placed in a petti dish and under the light conditions described previously. There was no discernible
response for a period of three weeks, at which time the seg*- ments were discarded.
Ten segments each of treatments 2, 3, and 4 which had not been under lig ht were dipped in Arasan and then put
into a germination chamber with constant light. Temperature varied between 33-37° C. One week later, a brown stain was evident around each segment. No further response was noted, and the segments were discarded afte r three weeks.
Experiment III
In an attempt to simulate natural conditions in
Tucson, Arizona, in late June and early July, f r u it segments 10 of the fol lowing types of Kal1stroem?a were pi anted in ten inch pots in sand in an Insulated walk-in chamber with con trolled light and temperature: Kailstroem?a sp. , depauperate;
K. cali forn ica, K. grand? flo ra , K, s£. from San Carlos Bay,
Mexico. Temperature varied between 23-25° C at night and between 42-45° C during the day. Although a lig h t meter was not available to measure the ;foot-cand1es, it was assumed that sufficient light was present for normal growth.. Pots were watered with tap water every other day.
The experiment was run for six weeks and then dis- continued, as no seeds had shown s igns of germi nation . When the fr u it segments were removed from the pots end examined, they showed no sign of water imbibition.
No attempts were made to replicate the above experi ments, for by early June seedling plants were located. These were transferred to the greenhouse.
Discussion
Neither refrigeration nor scarification with agita tion in rough sand appear to have an effect on germination.
If a chemical inhibitor is present, it would appear that it is not water soluble,'for no germination occurred after fruit segments had been washed in running water for twelve hours.
Tribu1us f r u it segments soaked in water in which KalIstroem?a segments had previously been soaked did not show a lower nor 11
a slower rate of germination than untreated Trjbulus fruit
segments.
It would appear that technique is the major problem.
If split fruit coat and extruded radicle are taken as an
indication of initiation of grpwth, then germination has been
achieved. However, a more precise control of conditions after'
germination is needed before Kal1stroem?a can be raised to maturity. ; : f ;■ ' CYTOLOGICAL STUDIES
A number of the genera in the Zygophy11aceae have been examined cytologically, and th eir chromosome numbers given in Darlington (5, 6), Schnack and Covas (16) found that Tribulus terrestris had 2n=48, while Sugiura (19) and
Negodi (12) found 2n=s24. An examination of root tips of
T» te rre s tris collected in Tucson, Arizona, showed a 2n num ber of 24.
Buds of Kal1stroemi a grand ? f 1 ofa were k ille d and
fixed in Karpechenko's fix a tiv e (14) over a period of two growing seasons at d iffe re n t times of day, from 6:30 A.M.
to 10:30 P.M. Anthers were removed from the buds and either stained with aceto-cdrmine and examined under a standard
lig h t microscope, or squashed in d is tille d water and examined using a phase microscope. To date, no meiotic figures have been found.
12 CHROMATOGRAPHY STUDIES
Paper chromatography has been used as a taxonomic tool by workers for a number of years. One of the most lucid examples of success using this technique is the work of
Turner and Alston (20) on species and hybrids of the Genus
Bapti s ia of the Leguminosae.
Two-directional paper chromatography was investigated as a means of separating the species of Kal1stroem?a and comparing them with Tribulus terrestris. A pigment-free extract of leaf material collected by the author (duplicate specimens are in the University of Arizona herbarium) was obtained by grinding leaves with sufficient absolute alcohol to equal 80% alcohol by volume. The insoluble material was removed by filtr a tio n using a Buchner funnel and washed with
80% alcohol. Three volumes of chloroform per volume of extract were added, and the mixture was shaken in a separa tory funnel (3). After separation, the aqueous layer (upper) was retained. Sample series of 1, 2, and 3 lambda were run.
Since there was no apparent difference between 2 and 3 lambda
2 were used in the fin al runs. Two lambda of each sample were spotted on Whatman number 1 Chromatography paper. Sol vent one consisted of 4 parts butanol: 1 part glacial acetic
13 . 14 acid; 4 parts water. The second solvent used was 4.0 parts methanol; 100 parts water: 2 parts pyridine. After the second direction had been run, chromatograms were dr fed and examined for v is ib le and fluorescent spots. They were then sprayed with 0.5% ninhydrin, dried at 46° C, and examined for ninhydrin-positive spots.
Chromatograms were run in two series, with a month's time between the series. Upon examination of the two series,
it was concluded that either because of the change in amino acid content during that period or because of differences in technique, the two series could not be compared. The samples of the Mt. Lemmon Ka11stroemia species were collected just prior to the second runs, and since results were very similar to the other Kalistroemfa spec?es run at that time (all of which had been collected before the first run), the differ ence between the two series of runs is probably due to d if
ferent conditions at the time of the runs, rather than to any change in amino acids In the extracts. This occurred even though attempts were made to minimize variables in the tech nique.
One specimen which had not been id en tified was run to determine if it could be correctly identified on a basis of the chromatograms alone. It was designated "Mt. Lemmon," the area in which it had been collected. A total of thirty-five 15
chromatograms were run. The f ir s t series of eight was not
used in the fin al comparisons. As species showed consistency
in spots in the various runs, only one set is included in
this thesis as an example. R f, or the ra tio of the solvent
front to the distance traveled by various compounds, is
plotted on the axes of the charts shown. Ninhydrin-positive
spots are marked With a c irc le , and fluorescent spots with a
small trian g le. Unless otherwise labeled, the ninhydrin*
positive spots were purple in color, and the fluorescent spots were blue.
On a basis of the chromatograms run, K. qran diflora,
K. parviflora and K. callfornica cannot be distinguished.
K. hirsutissima differs from the other three species in all
runs in number and placing of spots. This might correlate with the observation that K. hirsutissima. is toxic to
cattle (1). Tribulus terrestris is strikingly similar to
the Kal1stroemla species run, and would not be placed into
another genus using these chromatograms as a c rite rio n . The
Mt. Lemmon species, if identified using only the chromato
grams, could be ei ther K. qrandi f lo ra , K. p a rv iflo ra , or
K. cal?fornica.
Embryos used for one-directional chromatbgrams were
obtained by cracking fruit segments with pliers. They were
homogenized in a bacterial cell homogenizer with 5 cc of 16
1.0
RF Sc CO A J OVrcct* l*o A ,70
.UO
.20
.10
.10 .20 .30 .40 ,aO .UO .70 .to .^0 R F F; rsT D i rcc.Vx on
FIG. 1. Collected in the Mt. Lemmon area, a small-flowered specimen, but not keyed to species before chromatograms were made. Ninhydrin-positive spots are circled. The small "B" indicates a blue color, rather than the more com mon purple color.
Ria 'a C 17
i.o
.10 Q6
.20 .
.\0
IO .20 30 .4 0 . SO .1,0 .7 0 .VO .90 1.0 ^ f- F* v r D i f i o
FIG. 2. Growing in a yard in Tucson, Arizona.
Tr IdixIu S tc Vfcstn & YL
R un C. 18
.So
.10
10 2.0 .30 .WO .sTO .UQ> .10 .%Q .40 1.0 R F F i" rsT Di rcct-1 Ox>
FIG. 3. Growing in association with K. cal ? fornica and Tribulus te rres tr i s. Storm Collection number 160.
Run A 19
•8o
.30
AO .ZO . 30 M O .SO M o .70 . ?0 40 1.0
R F hi rsT D1
FIG. 4. Growing by its e lf as a roadside weed.
a. 11 sT rocw i Ok. <^rad.1 (- lofO.
R ul o CL 20
O
.10 . LO .30 M o .SO '(,0 MO .to .90 i.O R F t~ I'rsT Di‘rec T ton
FIG. 5. Associated with K. grand i flo ra and Tr ? bu1 us terrestr is. Fluorescent spots are marked with a trIangle. The smal1 "P" indicates a purple fluorescence, rather than the typical blue color. Storm Collection number 159.
Koill si'f ocvai a. ca! t^or aic a Ru a c 21
i.o
• (•0
Jo
JO .20 .30 .40 .aO .^O 70 .io .9o v o R F FiVat Dj/ccf :dv\
FIG. 6. Growing associated with K. grand i flo ra and Tribulus terrestris. Storm Collection number 152.
Ko-U s"^roe.>rv\ia. paru vVlorcx
^ .lo R F Second 80 . Dt re ct* ion • 16 ■ •30 • lo • ,10 o .10 • JO .wo .£0 • C.O .70 I . o RF F. rSt Dxrcc."V'vO'r> FIG. 7. Growing in association with K. grand ? flo ra and Tribulus terrestris. Storm Collection number 152. .20 .10 - I o 30 .40 S o .<*0 . "70 .9 0 .4 0 1.0 R F F rsT D. rcct.'orx FIG. 8. Growing by its e lf as a roadside weed. This shows the greatest number of spots. The "Y" indicates a yellow-colored ninhydrin-positive spot. Storm Collection number 156. 11 strocm i a Rul -n A d is tille d water. The suspensions were filte re d through a Hi 1i ipore H-R fi lter. To insure a high concentration of amino acids, 300 lambda were spotted on sheets of Whatman number 1 paper. Chromatograms were run using the descending method with a mixture of 4.parts butanol; 1 part glacial acetic acid: 4 parts water, When the solvent front had nearly reached the bottom of the paper, chromatograms were removed, a ir dried, sprayed with 0.5% ninhydrin, and placed in a drying oven at 50° C. After drying, they were examined for ninhydrin-positive* and fluorescent spots. Embryos which had been treated with low temperature, mechanical agitation , high temperature, and lig h t, as described in the previous germination experiments, were used. Fruit segments subjected to treatments 1, 2, 3, 4, and 8, K. cal i forn i.ca, K. ££. from Guaymas, Mexico, and Tr 1 bulus te r r e s tr is were run. The various treatments used appeared to have had Tittle effect upon amino acids of the embryos. Refrigeration may have some e ffe c t, as these embryos produced only one spot which differed in Rf from the other treated and untreated embryos run. One spot, at Rf 11, occurred in most treated and untreated species of KalIstroemia, and was not present in Tribulus terrestris nor in the three-year old embryos of K. grandif1 ora. One theory which attempts to account for the difficulty encountered in germinating Kallstroemia is that a long period of aging is required, during which a germination inhibitor is slowly deteriorated. This inhibitor, then, might be correlated with the spot at Rf IT. TribuTus, which lacks this spot, germinates very easily. However, fr u it seg ments three years old and untreated showed no greater activ ity than did any of the other segments used in the germination experiments. The two replicates of Tribulus differed. One of them had been stored overnight in the refrigerator, and had shown signs of growth before it was homogenized and run. This would account for the difference. Tribulus did d iffe r slightly from all the Kallstroemias, having one more spot at two Rf points away from one of the spots present in hi 1 the Kallstroemias. This is a relatively small difference, and could not be used as a basis for class?f(cation of Tribulus as being far different from the species of Kallstroemia. It would appear that on a basis of amino acid content of both leaves and embryos, Trlbu1 us terrestris is closely related to K. caljfornica, K. grand(flora, and K. parvif!ora. Unfor tunately, no embryos of K. hi rsutissima, which is distinct in chromatograms of leaves, were run. POLLEN VIABILITY Herbarium specimens collected by Al-Katib during the summer o f 1957 were examined to determine the per cent v ia bility of pollen grains of the four species of Kal1stroemia found in Arizona. It had been previously noted that K. cal ifo rn ?ca is almost exclusively found in association with K. grandi flora and Tribulus terrestr is. To test the hypothesis that Kal1stroemia califo rh ica might be the result of a cross between Tribulus and K. grand i flo r a » pollen grains from herbarium specimens were examined by staining with lacto-phenol cotton blue. Viable pollen stains a dark blue, and is spherical. Pollen grains thought to be inviable are either misshapen, much smaller than the normal, or stain a brown-black. One flower was removed from each herbarium sheet, and the anthers smashed directly in the 1acto-phenol Cotton blue. The variable number of grains counted resulted from the unequal number of herbarium sheets available. TABLE 2. Per cent viability of pollen of the four Arizona Kal1stroemia species Species No. grains counted Per cent viable K. grand!flora 20,899 99.3 K. californi ca kkO 90.5 K. hirsutissima 665 91.8 K. parviflora 1,341 95.8 26 27 As shown In Table 2, all taxa now called species in Ari zona show a hi gh degree of pol1en V ia b i1it y . One explana tion for this may be that the genomes of the four taxa and Trtbulus are similar enough so that hybridization, if it occurred, would not cause a decrease in pollen fe rtility. SUMMARY Since the time of Scopoli, Tribu10s and Kalistroemia have been separated on a basis of fruit structure. An attempt was made to find some other basis for comparison of the two genera, ancl chromatography, cyto lo g ical, and ppl len vlabil ity studies were chosen. Before these experiments were begun, a series of tests were run in an attempt to germinate seeds of Kailstroemia to provide a constant source of root tips, buds, and fresh material. Tribuius terrestfis germinates easily, but attempts to germinate and grow seeds of Kal1stroemia to maturity were not successful. However, radicle extrusion is an indication that germination can be effected. The major problem which remains is one of technique, rather than of finding conditions which will cause germination. Separation Of three of the four taxa of Kailstroemi a occurring in Arizona into species on a morphological basis is not substantiated by chromatograms of leaves or embryos. Chromatograms of Tribuius are simi1ar to Ka11stroemia grand!- flora, K. paryiflora, and K. ca1ifornica, and do not provide a basis for the separation of Tribuius into another genus. Chromatograms of leaves of Kal1stroemia hi rsutissima show unexplained but consistent differences when compared with 28 29 K„ qrandiflora, K. parviflora, K, cal?fornica, and Tribulus te r r e s tr !s'. As hfrsuti-.ss.fma is the only taxon known to be toxic to livestock, this property might be correlated with the difference in chromatograms. Examination of pollen grains for v ia b ility shows that the four taxa of Kallstroemia all possess a high viability. Unfortunately, this neither supports nor detracts from the hypothesis that some of the taxa now called species are the result of hybridization. Germination is a necessary prerequisite to karyotype and hybridization studies of the four taxa of Kallstroemia and of Tribulus. These studies should provide more definite clues to the exact relationships within the genera. As a result of the studies reported in this thesis, no evidence has been found to warrant separation of Tribulus and Kallstroemia into two genera. Until cytological and hybridization studies can be done, classification of the two genera must be based on morphological c rite ria . LITERATURE CITED 1. Al-Katib, Y. 1958. Comparative morphological study of the Arizona species of Kal1stroemia Scop. M. S. Thesis, University of Arizona, Tucson, Arizona. 2. Benson, L. l9o2. Plant taxonomy. Ronald Press Co., New York. 464 p. 3. Block, R. J. and K. W. Weiss. 1956. Amino acid hand book. Charles C. Thomas, Springfield, Illin o is . 386 p. 4. Brewer, W. H. and S. Watson. 1880. Botany of C alifornia. Vol. I. L ittle , Brown and Co. 628 p. 5. Darlington, C. D, and E. K. Janaki-Ammal. 194$. Chromosome atlas of cultivated plants. George Allen and Unwin, L td ., London. 397 p. 6. Darlington, C. D. and A. P. Wylie. 1955. Chromosome atlas of flowering plants. George Allen and Unwin, Ltd., London, 519 p. 7. Gray, A. 1852. PTantae Wrightianae. Part I. Smithsonian In stitu tio n , New York. 146 p. 8. Johnson, E. 1932. The puncture vine in California. Univ. of Calif. Coll. of Agr. Expt. Sta. Bull. 528. Univ. of CalIf. Printing Office, Berkeley, Calif. 42 p. 9. Kearney, T.H. and R. H. Peebles. 1960. Arizona flora. Univ. of C a lif. Press, Berkeley and Los Angeles. 1085 p. 10. Kurtz, E. B. 1960. Biochemistry of adaptation in plants to environment. The American Naturalist. XCIV(876); 237-243. , 11. Linnaeus, Carolus. 1753. Tribulus in Species pi anterum 1:386-387. 30 31 12. Negodi, G. 1939. Cariologia delle Rutaceae e d elle Zygophyl1aceae. Scientia Genetica 1:182-185. 13. Norton, J. B. S. 1898. Notes on some pi ants, c h ie f1y from the Southern United States. 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