PHYLOGENETIC AND BIOGEOGRAPHIC INFERENCES AS DRAWN FROM
REPRODUCTIVE ISOLATION WITHIN THE CHARA ASPERA
DETH. EX. WILLD. COMPLEX (CHAROPHYTA)
by
C. DOUGLAS CROY, B.S.
A THESIS
IN
BOTANY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
August, 1979
ACKNOWLEDGMENTS
I wish to express my sincere thanks to Dr. Vernon W. Proctor
for his guidance and unrelenting encouragement.
I also thank
Professors Michael C. Grant, John S. Mecham, and David K. Northington
for serving on my thesis committee.
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF TABLES
""v
LIST OF ILLUSTRATIONS
v
Chapter
I. INTRODUCTION
"i
II. MATERIALS AND METHODS
5
III. RESULTS
IT
IV. DISCUSSION
24
Present Relationships
24
Biogeographic and Phylogenetic Inferences
26
Addendum
30
REFERENCES
^^
APPENDIX A
^^
APPENDIX B
46
m
LIST OF TABLES
Table
Page
1.
Growth Areas and Light Intensities
2.
Clone Reference Numbers and Collection Sites
12
3.
Details of Crosses
^^
IV
7
LIST OF ILLUSTRATIONS
Figure
Page
1. Attempted Crosses (North American)
14
2.
15
Attempted Crosses (European)
3. Attempted Crosses (North American-European)
16
4.
^7
Attempted Crosses (North American-European)
CHAPTER I
INTRODUCTION
Over the past twenty years much has been written about the
biological species concept.
According to this concept, "species are
groups of interbreeding natural populations that are reproductively
isolated from other such groups" (Mayr, 1970).
Although Levin (1979)
has questioned the validity of the biological species concept, many
zoologists and vascular plant taxonomists have applied it as advocated by Dobzhansky (1951) and Mayr (1970).
With corroborating
data from a host of scientific disciplines, biologists have constructed evolutionary pathways for many taxonomic groups of vascular
plants.
Unfortunately phycologists have rarely employed this con-
cept because of the difficulties of performing controlled interbreeding experiments with algae (Griffin & Proctor, 1964; Proctor,
1975).
Problems frequently encountered are those associated with
establishing cultures of sexually reproducing forms, emasculating
monoecious forms, and mechanically manipulating thalli (Proctor
^
al_., 1971; Grant & Proctor, 1972).
Moreover, the absence of
obvious morphological markers on microscopic, homothallic algae has
similarly reduced the ranks of algae suitable for experimentation
(Proctor, 1971).
Thus the elucidation of genetic and evolutionary
relationships within freshwater algae has only marginally left the
starting line.
Plants of the algal genus Chara, commonly known as stoneworts
or muskgrass, however do not present these encumbrances.
They are
equisetoid in appearance, possess shoots from 5 cm to 2 m long, and
bear large external gametangia enclosed within jackets of sterile
cells.
All members of the genus Chara appear to be haplobiontic.
Sperm released into the water by antheridia fertilize egg cells
contained individually within oogonia.
Fertilization is indicated
by a change in color of the oogonium from red or green to black.
Oogonia not fertilized within a few weeks of reaching maturity bleach
and abort.
The blackened oogonium, henceforth termed an oospore,
eventually dehisces from the plant thallus and lands in the substratum where meiosis occurs with only one haploid product surviving.
The haploid product gives rise to a protonemal filament and the
mature plant soon develops as a lateral branch of this filament.
Capitalizing upon the ease with which fertilization can be
verified. Proctor (Proctor, 1970, 1971, 1975; Proctor and Wiman,
1971; Proctor et^ il-, 1971), Grant and Proctor (1972), and others
(MacDonald & Hotchkiss, 1956; Griffin & Proctor, 1964) have shown
that experimental breeding is an effective and reproducible technique
for explicating genetic relationships among charophyte species.
Results from breeding experiments indicate that although consistent
morphological discontinuities generally signal reproductive incompatibility, reproductive barriers may not be correlated w U h
morphological, cytological, or geographical characteristics (Proctor,
1975).
Synthesizing these observations. Proctor (in press) nas
3
proposed that:
1) populations of unisexual Chara plants on disjunct
landmasses are highly endemic, 2) such populations have long been so
genetically distinct that they are incapable of interbreeding—hence
long ago eliminating gene flow between continents, and 3) given the
previous two assumptions, one can read evolutionary sequences among
unisexual charophytes by examining the separation of primordial landmasses in conjunction with biogeographic, morphological, and cytological data.
Populations assignable to Chara aspera Detharding ex Willdenow
(=Chara globularis var. aspera Wood) range throughout northcentral
and northeastern North America as well as throughout Europe
(Corillion, 1957).
C_. aspera ranges in North America northward from
Cuatro Ci^negas de Carranza, Coahuila, Mexico (personal collection)
to Long Lake, Saskatchewan, Canada (Robinson, 1906).
It extends
west to at least the Lac de Bois Range, Kamloops, British Columbia
(Allen, 1951) and eastward to Long Island, New York (Wood & Imahori,
1965) and Vermont (Robinson, 1906).
In the southeast United States
it has been reported from Currituck County, North Carolina
(Choudhary & Wood, 1973).
In Europe similar morphotypes are found
from Lebrija, Sevilla, Spain (Proctor, personal communication) to a
northern limit at 68-70°N in Norway (Langangen, 1974).
C_. aspera
has been verified as ranging eastward in Europe at least to
Swinoujscie, N.W. Szczecin Prov., Poland (Migula, 1897).
There have
also been reports of C_. aspera in .Morocco, Algeria, and Tunisia
within Africa (Feldman, 1946).
4
The interfertility of these populations would not only
necessitate a reevaluation of Proctor's reasoning but would substantiate Wood's 1965 assertion (Wood & Imahori, 1965) that "on the
basis of geographic distribution of the species, little can be concluded concerning the phylogeny within the genus [Chara]".
This
thesis constitutes a report of research conducted to test the null
hypothesis that C^. aspera populations from North America and Europe
are interfertile.
Similarly the discovery of reproductive barriers
between populations within North America and within Europe will be
discussed, as will be the phylogenetic implications of the inabilities of £. aspera populations to interbreed.
CHAPTER II
MATERIALS AND METHODS
C.. aspera plants were collected from the Netherlands, France,
the United Kingdom, and Spain within Europe and from the states of
Wyoming, Colorado, New Mexico, and Coahuila within North America.
The exact collection sites as well as environmental descriptions are
listed in Appendix A.
As is evident from Appendix A, the habitats
varied greatly, ranging from peat bogs to oligotrophic lakes, from
ponds of elevations greater than 1600 m to coastal ponds, from
roadside ditches to lakes with hundreds of hectares of surface area.
The plants were cloned and cultured in glass iced tea jars.
Clones were raised in unsterilized soil obtained at a depth of 1.8 m
from a building excavation in Lubbock, Texas.
Sterilized red sandy
loam topsoil from a cotton field north of Lubbock was also frequently
utilized.
Appendix B.
For a full description of these soil types, refer to
Ostracods, cladocerans, and snails of the genus Helisoma
were added to each jar to control contamination by filamentous green
algae.
No attempt was made to keep the jars unialgal.
To achieve different regimes of light and temperature, the
plants were grown in four different locations:
outdoors, shaded
greenhouse, growth chamber, and laboratory lightroom.
Uninter-
rupted illumination was provided in the laboratory lightroom by
cool white fluorescent lights.
25 C.
The temperature was a constant
A daylength of 12 hours was maintained in the growth chamber
at a temperature of 21 C.
Daylight was extended within the green-
house to approximately 18 hours by banks of 150 W flood lamps
triggered by automatic timers.
In the greenhouse temperatures
fluctuated from 6°C in the winter to 35°C in the summer.
While
outside clones were exposed to daylengths ranging from 13 to 15
hours with an average daytime temperature of 28°C and an average
nighttime temperature of 18°C.
Ranges of light intensity in the
growth areas are listed in Table 1.
At any given time about one-half of the clones were in the
greenhouse with the remainder either in the lightroom, growth
chamber, or outside.
Although clones were reared in the greenhouse
and lightroom beginning May 1978, clones were not outside until
May 1979.
Clones that produced numerous gametangia in the greenhouse
or lightroom were not moved; clones not producing gametangia were
moved to the other locations in hope that the different conditions
would stimulate gametangia formation.
Lack of material with numerous
gametangia, particularly oogonia, was the principle factor that
limited the number of crosses attempted and in many instances
limited the number of oospores harvested.
In attempts to further induce gametangia, the environments
within the jars were substantially modified.
To jars containing
£. aspera that had not produced gametangia since collection, CaCO^
was added.
Similarly, about .01 g of aquarium salt was initially
7
TABLE 1
GROWTH AREAS AND LIGHT INTENSITIES
Roof
Greenhouse,
full sun
Greenhouse,
shade
Growth Chamber
Lightroom
43.2 klux
22.0 klux
2.0 klux
.48 klux
.66 klux
46.0 klux
15.0 klux
3.0 klux
.98 klux
.40 klux
13.0 klux
2.9 klux
.34 klux
.34 klux
44.6 klux
Avg.
16.7 klux
Avg.
2.6 klux
Avg.
.73 klux
Avg.
.44 klux
Avg.
3
added to many jars having volumes of about 900 ml.
Salt concentra-
tions as great as that of full sea water were also occasionally
employed.
Additionally, air was bubbled through several jars for a
period of one month and, to increase the amount of surface area of
water available for CO2 dissolution, plants were also grown in
aquaria and styrofoam ice chests.
had a pH between 8.0 and 9.0.
The water used in all experiments
Although salinities of two-third's
sea water and full sea water both resulted in the death of the
C_. aspera, the plants easily survived a salinity equal to one-third
sea water.
As based only on subjective data, the other modifications
dramatically influenced neither gametangia production nor plant
growth.
Future experimenters should start their investigations with
small jars of £. aspera placed under high illumination but moderate
to low temperatures.
Larger jars should be progressively filled by
periodic transplanting and subculturing.
However this subculturing
should not be done in winter for the plants take too long to recover
in spring.
Additional advice for future investigators includes:
1) don't be too eager to start crosses; a three litre jar filled
with female clone is a minimum requirement for each attempted cross,
and 2) productive experimentation with illumination and temperature
requires growth facilities that will dependably function for
several months.
In the current investigator's experimentation, crosses were
conducted each week using those plants possessing gametangia.
As a
means of verifying identity, clusters of antheridia were harvested,
preserved in 3:1 ethanol:glacial acetic acid solution, and were
later used to obtain chromosome counts.
Early crosses were
initiated by transferring both male and female plants to a new common container.
Later crosses (the majority) were established by
planting five to ten shoots of clonal male C_. aspera bearing
antheridia in the female's jar.
Observations were made weekly on
the presence of antheridia, oogonia, and oospores.
All oospores
observed were immediately collected and stored in vials containing
water from the cross container.
When either antheridia or oogonia
first became absent in the cross container, the cross was described
as having undergone a "period of simultaneously present gametangia"—
a period when the oogonia could have been fertilized.
Likewise, if
both antheridia and oogonia were present for one month, the first
"period of simultaneously present gametangia" was recorded; if
another month elapsed and antheridia and oogonia were still present,
a second "period of simultaneously present gametangia" was recorded.
Once established, data on each attempted cross was recorded until
each attempted cross was cancelled either because of the death of
one of the clones or because contamination made continued observation impossible.
Collected oospores were removed from the vials of water in
which they were stored and allowed to air dry in the dark at 25 C
in open Petri dishes for three weeks prior to germination.
Next,
the oospores were innoculated into glass jars filled with distilled
10
water and sandy loam farm soil obtained from a farm on University
Avenue, two miles north of Loop 287 in Lubbock, Texas.
These jars
were placed in indirect light within the laboratory lightroom at an
illumination of approximately .02 klux.
The combination of farm
soil and lightroom illumination was judged by previous preliminary
germination experiments to yield greater germination percentages than
had the following soil mixtures when placed in either the lightroom
or greenhouse:
farm soil + salt, farm soil + CaCO^, excavation soil,
excavation soil + salt, excavation soil + CaC02, sand, sand + salt,
and sand + CaCO,.
In the final germination experiments, observa-
tions were made weekly en the number of resulting germlings and the
vegetative appearances of these germlings were also described.
CHAPTER III
RESULTS
During this investigation striking differences in the C^. aspera
clones became apparent.
are listed in Table 2.
Clone reference numbers and collection sites
Those from Spain (X-303) and France (X-711)
set gametangia freely whereas those from New Mexico (711, 712) and
the United Kingdom (X-341 and X-715) never produced gametangia.
Clones from Coahuila, Colorado, Wyoming, the Netherlands, as well
as clone 546 from New Mexico produced intermediate numbers of
gametangia.
All clones, except X-715 from the United Kingdom, flourished
vegetatively.
Even in the summer when most clones seemed to grow
best, X-715 grew sluggishly and was difficult to propogate by subculturing.
Nearly all clones turned white and brittle during the
winter with only a few shoots surviving.
These results not only
suggest that the life cycle of C_. aspera is strongly regulated by
photoperiod, intensity of illumination, and temperature, but also
suggest that clones from different localities vary widely in their
habitat requirements for optimal growth and sexual reproduction.
Oospore production between attempted North American crosses
is summarized in Figure 1.
In Figures 1,2, 3, and 4, a solid
blackened circle indicates that oospores were produced; an empty
11
12
TABLE 2
CLONE REFERENCE NUMBERS AND COLLECTION SITES
546
both sexes available
Black Lake, Colfax County,
New Mexico, U.S.A.
711
only female available
Bitterlakes National Wildlife Refuge,
Chavez County, New Mexico, U.S.A.
712
only female available
Blue Hole pond, Guadalupe County,
New Mexico, U.S.A.
X-013
only female available
Cuatro Cienegas de Carranza,
Coahuila, Mexico
X-303
both sexes available
Lebrija, Sevilla Province,
Spain
X-341
only male available
Hayle Kimbro, Cornwall,
U.K.
X-701 (=Gnll42)
both sexes available
Seven Mile Lake, Albany County,
Wyoming, U.S.A.
X-702
both sexes available
Meebour Lake, Albany County,
Wyoming, U.S.A.
X-703
both sexes available
Gelatte Lake, Albany County,
Wyoming, U.S.A.
X-704 (=Gn974=Gn972). . . . Flatirons Gravel Co. Pond,
both sexes available
Boulder County, Colorado, U.S.A.
X_705
both sexes available
Coot Lake Pond, Boulder County,
Colorado, U.S.A.
X-706 (=Gnl057)
both sexes available
Steele's Pond, Boulder County,
Colorado, U.S.A.
X_711
both sexes available
Montpellier, Herault Departmeht,
France
X.7-12
both sexes available
Amsterdam, North Holland Province,
Netherlands
13
TABLE 2—Continued
X-713
both sexes available
Amsterdam, North Holland Province,
Netherlands
X-715
only male available
Mai ham Tarn, Yorkshire,
U.K.
X-717
both sexes available
Flatirons Paving Co. Pond, Boulder
County, Colorado, U.S.A.
14
Male
X
r—
O
•
C\J
O
•
r^ o
r^ o
X 3
x:s
en
o
•
r^o
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Wyo.
X-703
Wyo.
e
o o o
X-704
Colo.
X-705
Colo.
e e
f—
E
X-706
Colo.
o
X-717
Colo.
o
546
New Mex,
711
New Mex.
o
712
New Mex,
X-013
Mex.
o
Figure 1.
O X
I <D
xs:
O
Q
r - CU
Attempted Crosses (North American)
15
Male
•
CM
r— xz
r>.+j
l O )
xz:
X-712
CO
•
I— xz
r^4->
1 0 )
xz:
I—
un
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fOi«i:
I—
p^i.^
xz3
xiu
I *
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xcy)
\
P
I C L
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X-713
Neth.
o
X-341
U.K.
n3
E
X-715
U.K.
X-711
Fr.
•
X-303
Spain
o
Figure 2.
c
o-«en <t3
^
Neth. ©
<u
CO
I—
r>s..
o
Attempted Crosses (European)
16
Male
CNJ •
>— XZ
r-^ 4->
1 <u
x:
3
X-701
Wyo.
X-702
Wyo.
CO •
1— xz
t
X
0
<v
-z.
LO
CO 'i>^
1
X :D
1
X
ID
X
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O •.CO fO
1 a.
X oo
O
0
O
X-704
Colo.
0
0
X-705
Colo.
0
0
X-706
Colo.
0
X-717
Colo.
0
X-703
Wyo.
E
0
o
546
New Mex.
711
New Mex.
712
New Mex.
X-013
Mex.
Figure 3.
•
0
Attempted Crosses (North American-European)
17
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18
circle indicates that the cross was attempted but oospores did not
result.
Oospores were produced by the following combinations:
(female/male); (Wyoming/Colorado), (Colorado/Wyoming), (Colorado/
Colorado), and (Coahuila/Colorado). Oospores were not produced by
four combinations:
(Wyoming/New Mexico), (New Mexico/Colorado),
(Colorado/Colorado), and (Coahuila/Colarado). Apparent discrepencies
are explained by two observations:
1) Although other Colorado clones
may have hybridized to produce oospores, X-717 and X-705 did not,
and 2) Whereas Coloardo clone X-706 crossed with X-013 of Coahuila
to yield oospores, X-704 from Colorado did not.
Within Europe—refer to Figure 2—three combinations yielded
oospores:
Spain).
(Netherlands/Netherlands), (France/France), and (Spain/
The attempted crosses which did not produce oospores were
(Spain/France), (France/Spain), (Netherlands/France) and (Netherlands/Spain) .
As shown in Figures 3 and 4, the only attempted intercontinental
crosses that produced oospores were (Mexico/France), (France/
Wyoming), and (France/Colorado).
However no hybrid oospores germinated.
sults are listed in Table 3.
The germination re-
Under the heading of "Gametangia"
in the third column of Table 3 are several numbers in parentheses.
Each of these sets of parentheses represents one attempted cross in
one jar.
The number inside a given set of parentneses indicates
the number of times gametangia of both sexes were simultaneously
present within the jar; for example, (3) indicates that antheridia
19
TABLE 3
DETAILS OF CROSSES
Female
Male
Gametangia
546
X-303
New Mex. Spain
(1)(1)
X-717
546
New Mex. Colo.
(1)(1)
X-704
711
New Mex. Colo.
(1)
Percent
Germina0 OSpores
tion
X-013
Mex.
X-303
Spain
(1)(1)(1)
(1)
X-013
Mex.
X-704
Colo.
(1)
X-013
Mex.
X-706
Colo.
(1)(1)(1)
(1)(1)(1)
5
0
X-013
Mex.
X-711
Fr.
(1)(1)(1)(1)
(1)(1)(2)
1
0
X-303
Spain
X-303
Spain
(1)(3)(3)
(4)(5)
877
4
X-303
Spain
546
New Mex.
(1)
X-303
Spain
X-701
Wyo.
(2)
X-303
Spain
X-702
Wyo.
(1)(1)(1)
X-303
Spain
X-704
Colo.
(1)(1)(2)
(3)
Description
10 died;
max. height=20 cm
20
TABLE 3--Continued
Female
Male
Gametangia
X-303
Spain
X-706
Colo.
(1)(1)(2)
C3)
X-303
Spain
X-711
Fr.
C5)
X-701
Wyo.
546
New Mex.
(1)
X-701
Wyo.
X-705
Colo.
(2)
X-701
Wyo.
X-711
Fr.
(1)
X-701
Wyo.
X-712
Neth.
(1)
X-701
Wyo.
X-713
Neth.
(1)
X-702
Wyo.
X-303
Spain
(1)
X-702
Wyo.
X-711
Fr.
(1)(1)(1)
X-703
Wyo.
X-706
Colo.
(1)(1)(3)
X-703
Wyo.
X-711
Fr.
(1)
X-704
Colo.
X-303
Spain
(5)(1)
X-704
Colo.
X-704
Colo.
(1){1)(1)
Percent
GerminaOospores
tion
3
0
76
21
3
0
Description
all healthy;
max. height=10 cm
21
TABLE 3--Continued
Female
Male
Gametangia
X-704
Colo.
X-705
Colo.
(2)(4)
X-704
Colo.
X-706
Colo.
(1)(1)(2)
(3)
X-704
Colo.
X-711
Fr.
(1)(1)(2)
(2)(3)(3)
X-704
Colo.
X-717
Colo.
(1)
X-705
Colo.
X-303
Spain
(1)
X-705
Colo.
X-701
Wyo.
(1)(5)
X-705
Colo.
X-702
Wyo.
X-705
Colo.
Percent
GerminaOospores
tion
Description
a l l healthy;
max. height=20 cm
141
16
19
0
101
4
a l l healthy;
max. height=l.5 cm
(1)(1)(1)
27
4
single p l a n t ;
.1 cm t a l l ; weak
X-704
Colo.
(5)(5)(6)
171
2
two died;
max. height=l.3 cm
X-705
Colo.
X-711
Fr.
(1)(1)(6)
X-706
Colo.
X-706
Colo.
(1)(1)
17
0
X-706
Colo.
X-711
Fr.
(2)(3)(4)
X-711
Fr.
X-303
Spain
(2)(6)(7)
X-711
Fr.
X-701
Wyo.
(1)
7
0
22
TABLE 3—Continued
Percent
Germi nation
Oospores
Female
Male
Gametangia
X-711
Fr.
X-704
Colo.
(1)(1)(2)
113
0
x-7n
(1)(1)(1)
(1)(2)(3)
299
0
Fr.
X-706
Colo.
X-711
Fr.
X-711
Fr.
(1)(2)
29
0
X-711
Fr.
X-717
Colo.
(1)
X-712
Neth.
X-711
Fr.
(1)
X-712
Neth.
X-712
Neth.
(2)
22
18
X-713
Neth.
X-303
Spain
(2)
X-713
Neth.
X-706
Colo.
(1)
X-717
Colo.
X-303
Spain
(1)(1)
X=717
Colo.
X-705
Colo.
(1)
X-717
Colo.
X-711
Fr.
(1)(3)
Description
1 died;
max. height=3 cm
23
and oogonia were simultaneously present within that jar on three
separate occasions.
In the fourth column is listed the total number
of oospores produced by the clones.
The percentage of these oospores
that germinated within one month of innoculation is given in the
fifth column.
In the last column is a short description of any
germlings that were produced.
As shown in Table 3, the only oospores
that germinated were from the combinations (Colorado/Colorado),
(Wyoming/Colorado), (Netherlands/Netherlands), and (Spain/Spain).
Figures 1-4 and Table 3 summarize five salient points.
1)
Numerous reproductive barriers exist among populations of C^. aspera.
These barriers, prohibiting either oospore formation or germination,
may be between populations within a single continent as well as
between populations from separate continents.
2) No European plants
successfully interbred with North American plants to produce viable
offspring.
3) As predicted by Proctor (1967) the oospore germination
percentages were low.
4) The population from Spain is reproductively
isolated not only from all North American populations tested but also
from all European populations tested.
5) French females consistently
produced oospores when fertilized by males from Wyoming and Colorado,
U.S.A.
However the resulting oospores failed to germinate.
CHAPTER IV
DISCUSSION
Present Relationships
As stated previously the lack of plants bearing gametangia
severely limited the number of crosses attempted.
The lack of
sexuality among clones is probably related to their generally high
rate of vegetative growth.
All clones possessed white bulbils up to
1 mm in diameter attached to their rhizoids.
Olsen (1944) and
Migula (1897) speculated that bulbils may aid the plants to overwinter and that new vegetative shoots may arise from the bulbils.
Indeed Migula emphatically stated:
"Diese Knollchen bleiben am
Leben, auch wenn die vegetativen Theile der Pflanze durth Frost oder
Austrocknung zerstort werden."
Webster (1924) stated:
Additionally Groves and Bullock-
"Chara aspera does not fruit very freely,"
a condition that they attributed to an efficient method of reproduction by bulbils.
That new vegetative shoots arise from bulbils
has never been directly observed by the current investigator.
According to the traditional taxonomic literature, £. aspera
is distinguished by its possession of three-ranked cortication,
stipulodes in two tiers, bulbils, and a haploid chromosome number
of fourteen.
similarities.
The investigations here reported have confirmed these
Given that the common possession of these
24
25
characteristics most probably indicates that the European and North
American populations are of monophyletic origin, the oospores produced by the French females become noteworthy.
These oospores
further indicate that the European and North American £. aspera
populations, although currently reproductively isolated, are
genetically related.
Indeed, as revealed in these experiments,
there are three levels of genetic isolation among C. aspera clones:
plants may be intersterile to the extent of oospore formation,
oospore germination, or may be interfertile and produce oospores
which do germinate.
Against the background that Chara species from different
taxonomic sections are generally morphologically distinct and always
reproductively isolated (Proctor, 1975; Proctor, personal communication), the question arises as to what taxonomic rank the reproductively isolated North American and European £. aspera populations
should be assigned.
In accordance with the definition of sibling
species proposed by Mayr (1970), each reproductively isolated unit
should be considered a sibling species.
This contention is further
buttressed by Grant's (1971) definition of sibling species:
The ability of individuals to exchange genes successfully,
that is, to cross freely and produce fertile and viable
progeny, characterizes them as members of the same
biological species, whereas the inability to exchange
genes freely and successfully is the mark of separate
biological species. All else, including morphological
difference, is superstructure. . . . Traditional taxonomic
methods of classification and identification rest on this
common association of external morphological characters
with physiogenetic traits. But the correlation does not
always hold. We find cases of good biological species
which are virtually indistinguishable morphologically.
Such cryptic species are termed sibling species.
26
Furthermore, because the reproductive patterns within the £. aspera
complex parallel those found within recognized groups of sibling
species such as the Gilia transmontana group of herbaceous dicots
(Grant, 1971), the microalga Pandorina morum (Coleman, 1967), and
the Chara braunii Gmelin complex (Proctor, 1970), the North American
and European groups of C_. aspera should be regarded as assemblages
of sibling species.
Biogeographic and Phylogenetic Inferences
A paramount consideration is how and when reproductive barriers
arose within a presumably more uniform population that once extended
across at least parts of both Eurasia and North America.
The most
probable explanation would seem to involve both plate tectonics and
changing climates.
The unification of the two (or three) continental
landmasses within the supercontinent Pangaea (and later Laurasia)
has been well established and accepted (Dietz & Holden, 1970; Cain,
1971; Valentine & Moores, 1972; Sclater & Tapscott, 1979) as has the
general cooling of central and northern latitudes beginning with the
Cretaceous (Wolfe & Leopold, 1967; Wolfe, 1971; Wolfe, 1972;
Leopold & MacGinitie, 1972; Cox e_t al-, 1973; Cox, 1974).
Both the
opening of the Atlantic and the climatic cooling of North America
and Eurasia are here proposed as major events responsible for the
geographical distribution and speciation of £. aspera species.
These
events probably culminated in vicariant speciation, i.e. the production of a particular biotic distribution through the subd-/is1on
(vicariance) of an ancestral biota (Cain, 1944; Rosen. 1978).
27
Long distance dispersal is assumed to have played only a minor
role in either the distribution or speciation of C^. aspera species.
Cracraft (1975) has forcibly argued that, among alternative hypotheses
of vicariance and long distance dispersal, the former constitutes the
parsimonious choice.
Cain (1971) pronounced long distance dispersal
inadequate in itself as a theory.
Olsen (1944) suggested that
£. aspera is intolerant to sea water, a belief confirmed by experimentation performed during the course of these interbreeding
investigations.
The experimental salinity tolerances of C_. aspera
species agree favorably with those listed for C_. aspera by Olsen
(1944) and by Peck & Morales (1966).
sink even in salt water.
Furthermore, of course, oospores
Finally, Proctor (1975) has written:
The frequency and maximum distance that oospores are
carried by birds (not 'can be') remains unknown
but apparently dispersal seldom culminates in the
establishment of potentially Interfertile populations that extend over wide areas of the earth's
surface or even that of a single continent.
The reader may question why the flooding of the Bering Land
Bridge has not been included as a possible vicariant event.
The
primary reason is that the flooding did not occur sufficiently
early.
The Asian and Euro-American landmasses joined 140 million
years ago (Cox, 1974) producing the earliest land connection between
Siberia and North America.
The earliest flooding across the land-
bridge that redivided North America and Asia occurred in the
Pliocene (Hopkins, 1967; Einarsson et_ al., 1967), which began about
11 million years ago (Stebbins, 1966).
The rate of speciation among
28
charophytes has been regarded as slow (Grambast, 1974; SoulieMarsche, 1979), and Soulle-Marsche has traced the genus Chara back
to the Cretaceous period by comparisons of extant and fossil oospore
morphology.
Moreover the Chara connivens Braun and Chara austral is
Brown complexes serve as diverse estimates of speciation rates within
the genus Chara.
Species of C^. connivens from Spain, France, and
the United Kingdom are reproductively isolated from the other
C_, connivens species in Israel, Greece, and Romania (Proctor, 1975).
Proctor (personal communication) has suggested that the formation
of the Alps was the probable event which promoted the reproductive
isolation of the groups.
Assuming the major formation of the Alps
to have occurred in the Oligocene and Miocene (Bernoulli et al.,
1974), ending 11 million years ago (Stebbins, 1966), it appears that
reproductive isolation can evolve within 11 million years.
Yet the
reproductive barriers between C^. austral is populations on Australia
and India are still not absolute, and the postulated vicariant
event—the separation of the Indian subcontinent from Gondwanaland—
took place about 180 million years ago (Dietz & Holden, 1970).
Hence in another group 180 million years has not been sufficient
for absolute reproductive Isolation to develop.
Using these ages as
extreme boundaries, the origin of the reproductively isolated North
American and European C_. aspera assemblages could have occurred
during or between the Cretaceous period and the Miocene epoch—too
early to have been greatly influenced by even the first inundation
of the Bering Land Bridge.
29
The opening of the North Atlantic began with the separation of
Greenland and North America approximately 80 million years ago, and
most major topographical features were formed by 36 million years ago
(Sclater & Tapscott, 1979).
These ages fall well within the range of
dates ascribed to the origin of £. aspera species.
During approxi-
mately the same time there was a gradual climatic cooling across
northern latitudes of the Euro-American-Asian continental plates
(Wolfe & Leopold, 1967; Wolfe, 1971; Wolfe, 1972; Leopold & MacGinitie,
1972; Cox et_al_., 1973; Cox, 1974).
Cooling increased sharply in
the Miocene, set by Stebbins (1966) as beginning 25 million years ago,
with the greatest rate of cooling occurring later in the Pliocene
and resulting in the formation of glaciers during the Pleistocene
epoch (Cox et_al_., 1973).
This cooling probably gradually destroyed
northern £. aspera populations along both sides of the fledgling
North Atlantic so that eventually such populations became restricted
to more southern latitudes where the Middle Atlantic was much broader.
Therefore this climatic cooling, taken in conjunction with the formation of the Atlantic Ocean, should be considered a major vicariant
event.
According to Einarsson et_ a_l_., the ten glacial cycles which
occurred on the Tjornes peninsula of northern Iceland represent a
minimum estimate of the total number of world-wide Pleistocene cold
fluctuations and glacial advances.
Moreover, Einarsson et aj_.
(1967) have dated the first glaciation of the Tjornes peninsula as
having occurred between 1.9 and 3.0 million years ago.
While it is
extremely doubtful because of their recency that these glaciations
30
were directly responsible for the development of reproductive isolation between the North American and European C_. aspera complexes, it
is highly likely that they further fragmented the assemblages by
promoting the speciation of local populations within both landmasses.
That the formation of the Atlantic Ocean in conjunction with
the Cretaceous-Pleistocene cooling was the vicariant event which
produced groups of sibling C_. aspera species appears the ultimate
inference to be drawn from this interbreeding investigation.
At-
tempted crosses have revealed that the North American and European
C_. aspera species are reproductively isolated from one another, and
it seems unlikely that the inclusion of additional clones will result
in an appreciably different conclusion.
Such reproductive isolation
supports Proctor's (in press) contention that populations of unisexual Chara on disjunct landmasses are highly endemic and incapable
of interbreeding.
The described interbreeding experiments further
demonstrate that C_. aspera species differ in light and temperature
requirements as well as in their degree of gametangia production.
In conclusion, because C_. aspera species are highly endemic yet show
lines of common ancestry via their ability to form nonviable oospores,
plausible phylogenetic Inferences have here been drawn based on
geographic distributions-thereby directly contradicting Wood's
(1965) assertion that such cannot be done.
Addendum
To satisfy Popper's (1968) dictum that scientific explanations
differ from non-scientific ones only by virtue of their falsifiabil ^'ty,
31
in addition to supplemental evidence, means of falsifying the inferences that constitute much of this these are here provided:
1.
The discovery of a persistent, natural, and bisexual population
of £. aspera on a remote oceanic island would establish the validity
of long distance dispersal and thus obviate any consideration of
entrance to North America via the Bering Land Bridge.
2.
In addition to the areas already mentioned, iC. aspera is re-
portedly found in Spitsbergen, Italy, Greece, central and southern
Asia, and Newfoundland (Braun & Nordstedt, 1882; Groves & BullockWebster, 1924; Wood & Imahori, 1965).
However a collection from
Spitsbergen has subsequently been reclassified by Anders Langangen
as Chara canescens (Proctor, personal communication).
remaining areas should be verified.
Those from the
The discovery of C_. aspera
species in Greenland and Iceland and their confirmation in Newfoundland would, with the elimination of long distance dispersal, strengthen
the hypothesis that an ancestral population ranged across the landmass formed by their junction long ago.
Likewise, because of the
resulting increase in the verified range of C_. aspera species, additional support for the Laurasia-ancestral population hypothesis
would result from the confirmation of C_. aspera in Greece and Italy.
The discovery of C_. aspera groups in South America or Australia would
point toward either a common ancestral population on Pangaea—thus
greatly Increasing the estimated age of C_. aspera species — o r
toward long distance dispersal.
32
3.
The geographical locations and age estimates of fossil oospores
judged by multivariate techniques as most similar to current C_. aspera
oospores would be of tremendous value as potential falsifiers.
Platnick & Nelson (1978) have suggested that paleontology and geology
can be used to "falsify a correlation between a particular disjunction
and a particular barrier."
The discovery of fossil oospores judged
morphologically similar to existing C_. aspera oospores by a Bayesian
multivariate discriminant function analysis within strata laid down
before the opening of the Atlantic and climatic cooling had begun
would indicate that a common C_. aspera progenitor ranged across
Laurasia.
However such evidence depends upon two assumptions:
1) that
oospores from extant sibling species have changed little from those
of ancestral populations, and 2) that multivariate discriminant
functions can be used to accurately identify extant oospores.
Given
the postulated slow rate of evolution among charophytes, the first
assumption seems well founded.
The second assumption has yet to be
tested.
If it is discovered by statistical analysis that the extant
£. aspera sibling species can be easily differentiated from each other
on the basis of oospore morphology because of little or no overlap in
character range, then one may hypothesize that extant oospore
variability is possibly only a fraction of that which may have occurred within the ancestral populations.
If oospores assignable to
a specific extant sibling C_. aspera species are ever discovered
33
within strata that was laid down before the opening of the Atlantic
and climatic cooling had begun, then these events can be conscientiously dismissed as vicariant events because the extant
C_. aspera species, as delineated by oospore morphology, already
existed as separate morphological entities.
Clearly the analysis
of extant and fossil oospore morphology by multivariate morphometries should be placed high on the list of future studies.
4.
Lastly, if one can construct an accurate phylogeny of C_. aspera
sibling species and other Chara species by the methods discussed by
Hennig (1966), one should find other Chara species with close current relatives on opposite sides of the Atlantic, thereby corroborating the belief "that the distribution of our original group reflects
a general pattern of Interrelationships of areas and their history"
(Platnick & Nelson, 1978).
Multivariate morphometries as well as
paleontology and geology may be required to accomplish these tasks.
Further following the methods of Platnick and Nelson, if after
prolonged searching no other groups are identified which corroborate
the hypothesized relationships between C_. aspera species and the
tectonic movements of the European and North American continents,
the hypothesized relationships must be rejected.
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Mayr, E. (1970) Populations, Species, and Evolution.
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APPENDIX A
COLLECTION SITES AND HABITAT DESCRIPTIONS
In this appendix the exact collection site for each clone is
described.
These descriptions are the casual observations of the
principal collectors (Dr. Vernon W. Proctor, Texas Tech University;
Dr. Michael C. Grant, University of Colorado) and should not be
considered absolutely accurate environmental records.
and depths are estimates.
thesis for two reasons:
Temperatures
The descriptions are included in this
1) they enable other interested phycologists
to continue the interbreeding experiments here begun, and 2) by
demonstrating the great diversity of habitats occupied by C_. aspera
species, they not only abolish any contention that reproductively
Isolated groups are correlated along environmental gradients but also
demonstrate the difficulties encountered in establishing optimal
artificial environments for gametangia production.
The possibility
that a single C_. aspera species may be composed of myriads of
ecotypes has not been overlooked.
1.
546 Location:
Black Lake, Colfax County, New Mexico.
About
3.2 km from the community of Black Lake, New
Mexico.
Between 48 and 64 km from Eagle Nest,
New Mexico.
38
39
This 8 hectare lake reaches a depth of 3 m and a maximum summer
temperature of 25°C.
C_, aspera was collected from a mucky organic
bottom to a depth of ,3 m along all parts of the lake.
The lake,
located in a large hay meadow, was surrounded by peat and a large
amount of emergent vegetation.
2.
711 Location:
Bitterlakes National Wildlife Refuge, Roswell,
Chavez County, New Mexico.
33° 24'N).
(Roswell:
104° 33'W,
The collection site was in Unit 3
immediately above the dam.
The collection area consists of a black mud flat with a high organic
content that floods in February or March with brackish water and
usually dries by August.
At maximum size, the flooded area reaches
a depth of 1.2 m and covers about 60 hectares.
may reach 22 C in the summer.
to .2 m.
The C_. aspera was collected at depths
Additional Chara species observed include £. longifolia
and C_. hornemanii.
3,
The water temperature
712 Location:
Tamarix was abundant near the bank.
Santa Rose, Guadalupe County, New Mexico.
(104° 42'W, Gn 439 34° 56'N).
The C_. aspera was found in a fish hatchery pond 20 to 30 m from Blue
Hole Lake.
The maximum water temperature in the pond is about 15 C
in the summer.
The pond possesses an artificial sandy-gravel bottom
carpeted with C_. aspera and C_. contraria.
along its banks.
tinge.
Weeds surround the pond
The C_. aspera collected here has a reddish
40
4.
X-013 Location:
Pond, 19-24 km immediately south of Cuatro
Cienegas de Carranze, Coahuila, Mexico.
Cienegas de Carranza:
(Cuatro
104°W, 27°N).
The maximum depth of the ,04 hectare pond is about 3 m and in the
summer the water temperature may reach 30°C.
The C. aspera was
collected from a bottom substratum of calcareous marl at a depth of
.3 m.
£, aspera was very abundant though C^. contraria, C. hornemanii,
and £. zeylanica were also present.
5.
X-303 Location:
South of Lebrija, Sevilla Province, Spain.
(6
lO'W, 36
55'N).
Near junction of county
road C441 and the Cadiz-Sevilla railroad.
(Cadiz:
6° 18'W, 36° 32'N; Sevilla:
5°
24'w, 37° 24'N).
C_. aspera was collected from a 20-30 m wide ditch which was excavated
in a cattle pasture to create a railroad right-of-way.
The ditch is
several hundred meters long and contains brackish water reaching
a maximum depth of .75 m.
The ditch collects water only in the
rainy season and dries around the 1st of July and remains so until
autumn.
The maximum water temperature In the summer may range
between 25 C and 30 C.
C
The ditch possesses a heavy clay bottom.
aspera forms large mats in the ditch with the grass pasture
*
surrounding it.
6.
X-341 Location:
Shallow peat and mud bog with about .1 nectare
of surface area situated at Hayle Kinribro,
41
Cornwall, United Kingdom.
Hayle Kimbro is
located a few kilometers south of Helston,
Cornwall, United Kingdom.
(Helston:
5° 16'W,
50° 05'N).
The maximum water depth is .6 m and the maximum water temperature
in the summer may be 20°C.
The banks consist of a matrix composed
of tangled roots and peat with £. aspera growing in the cracks of
the matrix.
The prevalent Chara species were C_. aspera and C_.
globularis.
7.
X-701 Location:
Seven Mile Lake, Albany County, Wyoming.
Gnll42
(105° 45'W, 41° 15'N).
This pond of 1.2 hectare surface area reaches a maximum depth of
about 4 m and attains a maximum summer temperature near 21 C.
£. aspera was collected from the mud and gravel bottom substratum
at a depth of .3 m.
The C_. aspera was moderately abundant with
C_. contraria also present.
The lake is surrounded by sandy soil
covered with grass.
8.
X-702 Location:
Meebour Lake, Albany County, Wyoming.
Gnn53
(105° 51'W, 41° 08'N).
£. aspera was collected at a depth of .3 m out of the bottom substratum of sand and mud Interspersed.
The maximum depth of this
lake is about 1.2 m and in the summer the water temperature nay
range as high as 32 C.
observed.
£. aspera was rare.
C
longifolia was also
The lake, about 10 hectares in surface area, is surroundec
by sandy soil that supports mostly grasses.
42
9.
X-703 Location:
9 km west of Seven Mile Lake, Albany County,
Wyoming.
(Seven Mile Lake:
105° 45'W, 41°
15'N),
This lake of about 12 hectares surface area reaches a maximum depth
of about 9 m.
In the summer the water temperature may reach 30°C.
C_. aspera was collected at a depth of .4 m from a sand and gravel
(mostly sand) bottom substratum.
The C^. aspera was abundant and
C_, globularis and C_. contraria were also present.
The vegetation
around the banks consisted chiefly of grass with small quantities
of Carex and Typha present.
10.
X-704 Location:
Flatirons Gravel Co. Pond (105° 12'W, 40° 02'N)
Gn974
near Boulder, Boulder County, Colorado.
Gn972
This lake of .2 hectare surface area reaches a maximum depth of 1.2 m
and the water probably attains a temperature near 27 C in the summer.
C_. aspera was harvested from the mud interspersed with sand bottom
substratum at a depth of about .45 m.
C_. aspera was of moderate
abundance and C_. contraria, C^. evoluta, and C_. globularis were
likewise present.
Typha, Salix, Carex and assorted grasses were
observed along the bank.
11.
X-705 Location:
The soil surrounding the bank is clay.
A small pool (.13 hectare) immediately adjacent
to Coot Lake (8 hectares).
The exact location
near Boulder, Boulder County, Colorado is
105° 12'W, 40° 06'N.
43
The C_, aspera was collected at the maximum pool depth of .3 m out of
a bottom substratum consisting of mud and rocks.
the maximum water temperature may reach 27°C.
During the summer
C_. aspera was rare
in this pond with C_, contraria being much more abundant,
Carex
surrounded the pool which is located in a clay-like soil.
12.
X-706 Location:
Steele's Pond (105° 15'W, 40° 08'N) near
Gnl057
Boulder, Boulder County, Colorado.
From this .4 hectare, 1.8 m deep lake, C_. aspera was collected at
a depth of .3-,5 m.
The bottom was soft mud.
water temperature may reach 27 C.
C.. contraria were equally abundant.
The maximum summer
C_. aspera, C_. globularis, and
The lake is surrounded by
sandy soil supporting Carex and various grasses.
13. X-711 Location:
Coastal pond southwest of Montpellier, Herault
Dept., France.
(Montpellier:
3° 53'E, 43°
36'N).
This pond with about ,4 hectare surface area is located approximately
30 m from the coast.
The bottom consists of reduced, rotting mud.
C., aspera was collected in open water at a depth of 1.2 m.
The
prevalent Chara species was C_. aspera but Lamprothamnium and bluegreen algae were also found, thus indicating salt contamination from
the sea.
The pond was completely surrounded by Typha bogs.
14. X-712 Location:
Small roadside ditch, 1 m wide, located by the
Botshol nature preserve near Amsterdam, Nortn
Holland Province, Netherlands.
4° 54'E, 52° 2 r N ) .
(Amsterdam:
44
C_. aspera was the only Chara species present and was collected from
a bottom substratum of peat and sand at a depth of .6 m.
temperature may reach 19 C in the summer.
The water
The ditch itself possessed
banks bare of vegetation although it was surrounded by farmland.
15.
X-713 Location:
In Botshol nature preserve, about 8-16 km
from Amsterdam, North Holland Province,
Netherlands.
(Amsterdam:
4° 54'E, 52° 2 r N ) .
The lake is a twisted lagoon with about 400 hectares of surface
area lying at an elevation of only a few meters above sea level.
Although the lake reaches depths of 9-12 m, the C_. aspera was found
at a depth of only .6-.9 m.
near 19 C in the summer.
The maximum water temperature may be
C_. aspera was less abundant than C^.
globularis and C_. vulgaris.
The lake was entirely ringed by 6-9 m
of Scirpus and Typha with forest surrounding the Scirpus and Typha,
16.
X-715 Location:
Malham Tarn, Yorkshire, United Kingdom.
(exact position:
2° lO'W, 54° 05'N).
The lake is perfectly oval with no bays and reaches a depth probably
between 6 and 9 m.
C_. aspera was observed extending from a depth
of .15 m to 1.5 m.
The maximum summer water temperature is probably
near 18 C.
The bottom was covered with rocks the size of footballs
with the area between the rocks filled with sand from which the
C_. aspera grew.
C_. aspera was the dominant Chara species although
it was speculated that C_. globularis was probably also present.
,1
A cobbled beach extends down to the water's edge; further away,
the lake is surrounded by peat bogs.
17,
X-717 Location:
Flatirons Paving Company Pond (105° 16'W,
39
59'N) near Boulder, Boulder County,
Colorado.
This 6 hectare pond attains a maximum depth of about 6 m and a
maximum summer temperature near 27 C.
C^. aspera was collected from
the rocky, muddy bottom substratum at a depth of about ,3 m.
Besides the abundant C_. aspera, C_, globularis and £. contraria were
also found in the pond.
The pond was ringed by Carex, Typha,
grasses, and other common aquatic plants.
D
APPENDIX B
SOIL DESCRIPTIONS
Excavation Soil
Acuff series.
B25t horizon (1.5 m to 2.0 m depth)
Reddish yellow (5YR 6/6) sandy clay loam, yellowish red (SYR 4/6),
moist; weak coarse prismatic structure parting to weak medium
subangular block; hard, friable; few fine roots; common fine pores;
thin patchy clay films on ped surfaces; sand grains bridged and
coated with clay; common films and threads of calcium carbonate;
calcareous; moderately alkaline.
Farm Soil
Estacado series.
Ap horizon (0 m to .25 m depth)
Brown (7.5YR 4/2) clay loam, dark brown (7.7YR 3/2), moist; weak
medium granular structure; hard, friable; common fine pores;
calcareous; moderately alkaline; abrupt smooth boundary. .
Source:
Blackstock, D.A. (1979) Soil Survey of Lubbock County,
Texas. U.S. Department of Agriculture. Soil Conservation
Service. Texas Tech University Press, Lubbock, Tex.,
vili-105 pp., 65 pi.
46