Abstract:
The intertidal red alga Mastocarpus papillatus was sampled for genetic diversity
in three populations from central and southern California. M. papillatus can exhibit an
asexual apomictic life history, with female gametes giving rise to female gametophytes.
A higher proportion of this asexual reproduction occurs in populations in central
California and further north (Zupan and West, 1988). It is therefore hypothesized that
this geographic variation in life history should affect the population structure of M.
papillatus.
DNA was extracted from haploid female gametophytes and then fingerprinted
using the amplified fragment length polymorphism (AFLP) technique (Vos et al. 1995).
A bootstrapped consensus tree was constructed using parsimony. Localities were shown
to have distinct population fingerprints. Greater population structure was found in the
northern, more asexually reproducing populations, but this was most likely due to
sampling methods. No definite link was found between the life history and population
genetics.
Introduction:
Background
Mastocarpus papillatus is one of the most common red algae on the California
coast (Abbott and Hollenberg, 1976). It occurs in the mid to high intertidal and is often
dried completely during low tides. Classified in the family Petrocelidaceae, Mastocarpus
follows a typical red algal life history with a tetrasporophyte, gametophytes, and a
parasitic carposporophyte (figure 1).
Mastocarpus grows in a crustose form as a tetrasporophyte and in a foliose form
as a gametophyte. Male and female gametophytes are morphologically similar with
males lacking the papillae, which house the developing carposporophyte, and usually
showing less dichotymous branching than the females. In addition to the sexual cycle,
females can asexually reproduce in an apomictic cycle whereby unfertilized female
gametes mature in the papillae like carpospores but grow directly into female
gametophytes on release from the papillae.
The heteromorphic life history of Mastocarpus has been explained by Paine et al.
(1979) with the “bet-hedging hypothesis." Crusts are long-lived and an average sized
individual, 123 cm sq., can be as old as 87 years (Paine, 1979). But crusts are also poor
establishers. Gametophytes are mostly annuals but are well adapted for establishment
and long distance dispersal by the sea. The two morphologies occupy different ecological
niches and serve different purposes in the proliferation of the genus.
M. papillatus has a disjointed range in the Pacific. It occurs from Punta Baja in
Baja California north to the Aleutian islands in Alaska (figure 2), and as smaller
populations in Japan and Chile.
Zupan and West (1988) showed that the proportion of females with fertilized to
unfertilized carpospores varied between populations in the North America range. More
specifically, the proportion of sexually reproducing plants increases from nouth to sorth.
This trend in the proportion of sexual reproduction also occurs in M. stellatus on the
Atlantic coast of Europe (Guiry and West, 1983). Zupan and West (1988) also found that
all carpospores from a single female followed only one developmental cycle, either
apomictic or sexual.
Experimental Design
The purpose of this study was to link this aspect of the life history with population
genetics. To determine a phenogram for the populations studied and resolve the
population structure, amplified fragment length polymorphism (AFLP) (Vos et al. 1995
was used to give a genetic fingerprint of each individual studied. Culturing carpospores
from each blade fingerprinted would tie the reproductive cycle of the blade to its
fingerprint and thus tell how asexual and sexual reproduction influence population
genetics in M. papillatus.
It might be expected that the high rate of mixing in a sexually reproducing
population would lead to less isolation and therefore less genetic variability and less
population structure. Less population structure was found in the southern-most
population studied, but the collection methods were not documented by the collector and
therefore results are called into question.
Materials and Methods:
Collecting Samples
Asexual or sexual reproduction can be indicated by the culture of carpospores.
The female blades are the common phase in the two types of reproduction, making female
gametophytes ideal for sampling.
Zupan and West (1988) sampled from 8 sites in Baja California and California
from Punta Baja to Trinidad Head. I tried to approximate their sampling on a smaller
scale for this study.
Female gametophyte blades of M. papillatus were collected from three localities
on the California coast: the Santa Cruz small yacht harbor, Hopkins Marine Station in
Pacific Grove, and the University of California at Santa Barbara Marine Station (figure
3). Santa Cruz samples were collected from a stretch of shore approximately 6 meters
long, while at Hopkins Marine Station, the sampling was done over approximately 100
meters of shoreline. Samples 8, 9, and 10 from Hopkins came from rocks on the Fisher
beach (at one end of the 100m stretch) while samples 1, 2, 3, 5 and 6 came from rocks on
the Bird Rocks side of Agassiz beach (at the other end of the 100m stretch), and samples
4 and 7 came from the Dive Lockers side of Agassiz beach. The collection area for the
Santa Barbara samples is unknown because they were collected by someone else, and
methods were not documented.
Santa Cruz samples were dry at low tide when collected. They were stored at
-20°C for about one week and then thawed and rehydrated in seawater before DNA
extraction. Samples from Hopkins and Santa Barbara were stored wet at 4°C overnight
before DNA extraction. This storage appeared not to make any difference for DNA
content or quality as extractions from fresh blades produced similar results to extractions
from stored blades.
DNA Extraction
The following DNA extraction procedure was scaled down from a DNA extraction procedure from Jason Smith.
Blades were prepared for DNA extraction by first rinsing in double deionized
water and removing all colonizing organisms, then scraping off all papillae and the
cuticle with a fresh razor blade. The papillae contain the carposporophytes which are a
different generation in the alga’s life cycle. This tissue can contain DNA from a
fertilizing male and is therefore eliminated. Samples were then briefly washed in 10
percent hydrogen peroxide and blotted dry. From each cleaned blade were taken two 0.15
to 0.20 gram sections. These two sections were then treated as separate samples
throughout extraction, purification, and AFLP in order to act as controls for each other
over the rest of the protocol.
Each piece of blade was ground to fine powder in liquid nitrogen in a small
ceramic mortar and pestle which was cleaned with BACDOWNO DETERGENT
DISINFECTANT (Decon Labs, Inc), 70 percent ethanol, and 10 percent hydrogen
peroxide (3 minute soak) between samples.
The powder was then placed in a 1.5mL microcentrifuge tube. To each tube was
added 400uL 2X CTAB buffer (w/PVPP), 5uL 10 percent SDS, and luL B¬
mercaptoethanol. The tubes were then placed in a 55°C water bath for 10-12 minutes.
During the incubation, the tubes were vigorously shaken every 3-5 minutes.
After the incubation, 500uL of 24:1 chloroform:isoamyl alcohol was added to
each tube. The tubes were gently shaken for about 5 minutes then centrifuged for 20
minutes at 12,000 rpms in a microcentrifuge.
The red to colorless supernatant (usually about 300-400uL) was removed, taking
care not to transfer any particulate matter or chloroform (green), and placed in a new 2mL
centrifuge tube. If the supernatant was not clear, it was transferred to a new 1.5mL tube
and extracted with fresh 24:1 chloroform:isoamyl alcohol. Äfter centrifuging, this
supernatant was always clear. This supernatant (approx. 150uL) was then transferred to a
new 1.SmL tube for purification.
DNA Purification
DNA purification was carried out using a GENECLEAN II kit (Bio 101, Inc) with
the following specifications/modifications: 1OuL of GLASSMILK and Nal solution of
about three times the volume of supernatant was used per sample. Only two NEW
washes were performed. DNA was resuspended in two 15uL aliquots of double
deionized sterile water.
Amplified Fragment Length Polymorphism (AFLP)
The AFLP procedure by Vos et al. (1995) with modifications by DeTomaso et al.
(1998) requires the following steps: cut with restriction enzymes and ligate
oligonucleotide adapters, pre-AFLP PCR reaction, and AFLP reaction with radioactive
P-labeled primer (figure 4, from Tim Schaeffer)
Approximately 100ng of DNA per sample was used in each cut and ligation. The
cut reaction was carried out using 5 units each of Msel and EcoRl, from New England
Biolabs (NE), in a 30uL reaction with NE Buffer 2 for 1.5 hours at 37°C. The
oligonucleotide adapters were ligated on to the DNA fragments using 15 units of T4
ligase (NE) in a 40uL reaction for 10 hours at 37°C. Each cut and ligation was diluted up
to 100uL with TE (.1). 4uL of this solution was used in each 20uL pre-AFLP PCR
reaction (preamp).
Tnoticed in testing for the cutting ability of EcoRl and Msel on the purified DNA.
that EcoRl did a very poor job of cutting the DNA. While DNA cut with Msel appeared
as a smear on a 1 percent agarose gel with ethidium bromide, EcoRI cut DNA appeared
as the same sharp band that was originally produced by purification. Some EcoRI cutting
must have occurred for the AFLP to work. It is possible that there are methylated areas
on the DNA which block EcoRl cutting.
For the preamp, 30ng EcoRl (A selective) primer and 30ng Msel (T selective)
primer were used with 0.5 units of Taq DNA polymerase (PERKIN-ELMER). The pre¬
AFLP PCR was run in a PERKIN-ELMER model TCI thermocycler. A 72°C start for 2
minutes followed by 20 cycles of 94°C for 45s, 56°C for 45s, 72°C for 1min was carried
out. The completed reaction was diluted 1:20 with TE (.1).
3uL of the preamp DNA was used in a lOuL reaction for the AFLP PCR. The
AFLP PCR was carried out using a radioactive 32P-labeled EcoRl (ACT selective) primer
and a non-labeled Msel (TC selective) primer. The thermocycling was done in a Techne
GENIUS PCR machine with heated lid.
The AFLP PCR thermocycles were as follows: 95°C hot start, 2min; 94°C
denaturation, 45s; 65°C primer annealing, 45s; 72°C polymerization, Imin; the next 9
cycles were run as a step down function, decreasing the primer annealing temperature by
1.0°C each time until the primer annealing temperature was 56°C; at this step, the
program switched to a repetitive thermocycle of 94°C for 45s, 56°C for 45s, and 72°C for
1 min for 27 cycles.
The completed reaction was diluted with 2X loading dye to stop the reaction.
Samples were kept on ice over this period, then heated to 95°C for 4 minutes and put back
on ice. 4uL of each sample was then loaded in a 19 percent denaturing acrylamide gel
(38 x 50cm dimensions) with 42 percent urea and run for 2 hours and 40 minutes at a
constant 70W. The gel was then transferred to paper and dried for 1.5 hours with vacuum
and heat.
KÖDAK X-Omat film was then exposed on the gel for 12-24 hours and
developed in an automated developer.
Scoring and Analysis
19 bands were scored by eye for presence/absence of radioactive 33P-labeled PCR
fragments over the 30 total samples. Because the female gametophytes are haploid,
scoring a lane for presence/absence of bands gives a haplotype (Glenns, 1999). The
collected data was inputted as a matrix into a PAUP* program. A heuristic test for
parsimony was run 100 times. A strict consensus was taken from the 5 trees yielded. This
strict consensus tree was bootstrapped 500 times to yield the presented tree.
Results:
Figure 5 shows a section from the gel that was scored for the phenogram. Figure
6 shows the bootstrapped unrooted phenogram generated by PAUP*. All populations
cluster by locality as would be expected. Fingerprints from the Santa Barbara population
were noticeably different from Hopkins and Santa Cruz, and this showed up in the
phenogram.
Little population structure is seen within the Santa Barbara clade with only a 61
percent bootstrap value separating the two clades within the Santa Barbara population.
More structure is seen within the Santa Cruz and Hopkins clades. Note the bottom three
samples in the Hopkins clade. The 98 percent bootstrap value puts these samples clearly
in their own clade.
Discussion:
Tattribute the source of the population structure within localities to collection
methods. As mentioned earlier, in Santa Cruz I collected from a stretch of shoreline
about 6m in length, while at Hopkins I sampled over about 100m. That there is more
population structure at Hopkins is not necessarily due to the location. Samples from the
first Hopkins clade (1, 2, 3, 5, 6) came from the Bird Rocks side of Agassiz beach.
Samples 4 and 7 came from the other side of the beach, and samples 8, 9, and 10 came
from Fisher beach. The population structure seen in the Santa Cruz samples indicates
that population differences occur over very short distances in M. papillatus. The
population structure in the Hopkins clade also indicates small scale population
differences. From the population differences seen, it seems that the scale of variation in
Mastocarpus is quite small, so that a short stretch of sand between rocks could provide a
high degree of isolation. It would be interesting then to see if rocky areas have the same
degree of population structure as sandier areas.
It is also possible that the higher proportion of asexual reproduction contributes to
the population structure of the Santa Cruz and Hopkins clades. Asexual reproduction
could give more genetic isolation and cause the observed population structure. More
mixing of genes from the greater proportion of sexual reproduction in Santa Barbara
would give less isolation and then yield less population structure.
Carpospore cultures remain to be done. These would link the type of reproduction
in a female blade to its genetics. It could then be resolved whether asexual reproduction
in these plants allows for more population structure and how much mixing is going on
between sexually reproducing stocks. This would then help to explain why the species is
so dependent on asexual reproduction over so much of its range.
Many photosynthetic organisms in their home range have increased asexual
reproduction at the extremes of their range (Zupan 1999) and (Van den Hoek 1975).
The
pattern seen in Mastocarpus is not unique except that the proportion of asexually
reproducing individuals is extremely high over the vast majority of its range. That sexual
reproduction is so completely dominant in the far southern reaches of the M. papillatus
range could indicate that the species originated there. It seems then that the next step in
this study should be to root the tree using the closely related M. jardinii so that the basal
locality on the tree can be determined.
Acknowledgements:
Twant to thank Dennis Powers for his charismatic support and Tony DeTomaso,
Lab King, for taking me under his wing and sharing with me his mastery of all science. I
also want to thank Jason Smith for giving me his brilliant DNA extraction procedure. I
appreciate the time, thought, and support that I got from all of the following people: Ken
Callicott, Don Kohrs, Brad Magor, John Glenns, Tim Schaeffer, George Somero, William
Gilly, John Zupan, Judy Connor, and Rafe Sagarin.
References
Abbott, Isabella A. and George J. Hollenberg. 1976. Marine Algae of California.
Stanford University Press: Stanford, CA.
DeTomaso, Anthony W., Saito, Y., Ishizuka, Kathi J., Palmeri, K.J., and Weissman, Iry
L. 1998. Mapping the genome of a model Protochordate: I: a low-resolution
genetic-map encompassing the fusion/histocompatibility (Fu/Hc) locus of
Botryllus-schlosseri. Genetics 149: 277-287.
Glenns, John. 1999. from personal communication.
Guiry, M.D. and J.A. West. 1983. Life history and hybridization studies on Gigartina
stellata and Petrocelis cruenta (Rhodophyta) in the North Atlantic. Journal o
Phycology 19: 474-494.
Paine, R.T., C.J. Slocum, and D.O. Duggins. 1979. Growth and longevity in the crustose
red alga Petrocelis middendorffii. Marine Biology 51: 185-192.
Smith, Jason. 1999. from personal communication.
Schaeffer, Tim. 1998. from dissertation with permission.
Van den Hoek, C. 1975. Phytogeographic provinces along the coasts of the North
Atlantic ocean. Phycologia 14: 317-330.
Vos, Pieter, Rene Hogers, Marjo Bleeker, Martin Reijans, Theo van de Lee, Miranda
Hornes, Adrie Frijters, Jernia Pot, Johan Peleman, Martin Kuiper, and Marc
Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids
Research 23: 4407-4414.
Zupan, John R. 1999. from personal communication.
Zupan, John R. and John A. West. 1988. Geographic variation in the life history of
Mastocarpus papillatus (Rhodophyta). Journal of Phycology 24: 223-229.
Figure Legends
Figure 1: Life history of Mastocarpus papillatus. The sexual cycle proceeds from
the tetrasporophyte (2n). Meiotically produced tetraspores grow into male and female
gametophytes (n). The gametophytes mitotically produce gametes. The female gametes
are retained in the papillae. Male gametes are dispersed by wave action. Fertilization
occurs in the papillae on the surface of the female blade. The fertilization event produces
a carposporophyte (2n) which is parasitic on the female blade. The carposporophyte
mitotically produces carpospores which grow into tetrasporophytes on release.
The asexual cycle proceeds from the female gametophyte. Female gametes which
go unfertilized begin growing in the papillae in the manner of a carposporophyte. Spores
that are produced grow directly into female gametophytes. This is known as apogamy.
Figure 2: Map of the North American range of Mastocarpus papillatus, highlighted
in red.
Figure 3: Map of California indicating the three sites sampled in this study. Blue
arrows point to the sampling sites, indicated by red squares. SC indicates Santa Cruz site.
HMS indicates Hopkins Marine Station site. SB indicates Santa Barbara site.
Figure 4: Chart explaining the amplified fragment length polymorphism (Vos et al.
1995). Produced by Tim Schaeffer (1999)
Figure 5: Gel showing the banding patterns characteristic of the three sites. The 10
lanes on the left are Santa Barbara samples. The middle 10 lanes are Santa Cruz samples.
The remaining lanes on the right side are Hopkins samples. Bands that were scored are
indicated. Not all samples that were scored for the phenogram appear on this gel. Some
of the Hopkins lanes were not scored for the phenogram. Indicated sample numbers do
not correspond to those shown on the phenogram. A different autoradiogram was used
for scoring bands.
Figure 6: Parsimony tree based on scoring of bands on an autoradiogram
representing bands of 33P-labeled DNA fragments. Samples from Santa Barbara are
listed as SB 1 through SB 10. Samples from Santa Cruz are listed as SC 1 through SC
10. Samples from Hopkins Marine Station are listed as HMS 1 through HMS 10. Note
that the tree is unrooted.
119
Apogamy
A

—

A
foliose
female gametophyte
(n)
Tetraspoces —
melosis




crustose
tetrasporophyte
(2n)
Capospores
ce leased
P gametes
mitosi5
gametes
MtOSIS



S

foliose
male gametophyte
(n)


parasitic
carposporophyte
(2n)
fedilizaton
f 2










+




4
SC
HIS
5B —
—

4
AFL
Amplified Fragment Length Polymorphisms
(Vos et al. 1996 NAR 23: 4407)
AATTCNNNN..NNNNT
Digest HMW DNA with
GNNNN..NNNNAAT
ECORI and Msel
CTCGTAGACTGCGTACCAATTCNNNN..NNNNTTAACTCAGGACTCAT
Ligate ECORI and Msel
CATCTGACGCATGGTTAAGNNNN. .NNNNAATTGAGTCCTGAGTAGCAG
ite-spe adap
Selective Pre-amplification
GACTGCGTACCAATTCA
CTCGTAGACTGCGTACCAATTCNNNN. .NNNNTTAACTCAGGACTCAT
PCR with:
CATCTGACGCATGGTTAAGNNNN..NNNNAATTGAGTCCTGAGTAGCAG
ECORI XI MselY
AAATTGAGTCCTGAGTAG
Primers
Selective Fingerprinting
GACTGCGTACCAATTCAAGC
Amplification
CTCGTAGACTECGTACCAATTCANNN.. NNNTTTAACTCAGGACTCAT
CATCTGACGCATGGTTAAGTNNN..NNNAAATTGAGTCCTGAGTAGCAG
PCR with combinations of:
CTAAATTGAGTCCTGAGTAG
ECORI XnnnI Msel Ynnn
Primers
CTCGTAGACTGCGTACCAATTCAAGC NGATTTAACTCAGGACTCAT
Gel Analysis
)
CATCTGACGCATGGTTAAGTTCG..NCTAAATTGAGTCCTGAGTAGCAG
O
400 bp
1.5 kb
= =
= —
0.1 kb
— 100 bp
FN: aflpmth.cdr
58

15
6

(
9
EW
a
9 6
61

60
100
65
74
—
—
74
98

SE 1
SB 2
SB 3
SE 4
SB 5
SB 6
SB 7
SB 3
SB 9
SE 10
SC 1
SC 2
5C 7
SC 2
SC 10
SC 3
SC 4
sc 5
SC 6
5C 9
HIS 1
IIS 2
IES 3
HPIS 5
HIS 6
HIS 4
INIS 7
HPIS 8
HES ?
HPIS 10