Journal of Foraminiferal Research, v. 2 I , no.

2, p, 159- 167, April 1991

TOWARD A CLASSIFICATION OF PLANKTONIC FORAMINIFERA BASED ON

BIOCHEMICAL, GEOCHEMICAL, AND MORPHOLOGICAL CRITERIA
LISAL. ROBBINSAND NANCYHEALY-WILLIAMS~

organism because their amino acid sequence is encoded by DNA (the genotype). Genetic information may
be discerned from the protein make-up of the organic
matrix found in the CaCO, test of planktonic foraminifera (Abelson, 1955; Hare, 1969; King and Hare,
1972). Biochemical analyses by Robbins (1 987) and
Robbins and Brew (1990) have also shown that the
tests contain preserved proteins. This paper represents
a first attempt to integrate several new approaches for
obtaining genetic information from Recent planktonic
foraminifera.
Planktonic foraminifera, with their nearly continuous record of preservation in deep sea sediments, exhibit morphologic variations through time (Malmgren
and Kennett, 198I , 1982; Gary and Healy-Williams,
1987) and are ideal fossils for testing theories about
the tempo and mode of evolution. By analyzing discrete, preserved proteins and polypeptides from the
shells of fossil planktonic foraminifera and integrating
these data with quantitative morphology and stable
isotope data, we hope to clarify taxonomic distinctions.
The ultimate goal of this more sophisticated taxonomical approach is to help refine the biostratrigraphic
framework based on planktonic foraminifera.
Understanding the environmental parameters responsible for phenotypic variation of planktonic foraminifera is another area where biochemistry could
make an important contribution (Bolli, 1950; Kennett,
1968; Malmgren and Kennett, 1972; B6 and others,
1973, among others). The use of morphology as a paleoecologic indicator assumes that planktonic foraminifera are generally panmictic, Le., without isolated
populations. Recent work by Healy-Williams and others (1 985) and Healy-Williams ( 1 988) questions this
assumption by revealing significant stable oxygen and
carbon isotopic differences between morphotypes of
Globorotalia truncatulinoides and Neogloboquadrina
pachyderma. Recent work suggest that the morphotype effect may be the result of genetic differentiation
within populations, although this does not rule out the
possibility that the isotopes may be measuring environmental differencesas well (Healy-Williams and others, 1985; Robbins, 1987; Williams and others, 1988a).
Biochemical analysis of the morphotypes in terms of
protein differences could supply the necessary information to test the hypothesis that these populations
are discrete.

ABSTRACT

New data on the protein composition of the organic

matrix from tests of six species of planktonic foraminifera demonstrate that the amino acid composition of
single proteins from fossil shell matrices provides information on genetic relatedness. We present here preliminary results which utilize an integrated approach
to decipher intraspecific variation of planktonic foraminifera. Protein biochemical data from two morphotypes of Globigerinoides ruber provide an example of
how quantitative morphology, stable isotope analysis,
and biochemical analysis can provide the basis for an
integrated approach to foraminiferal taxonomy. This
combined approach could lead to a classification framework that more closely reflects genotypic relationships
than does the standard morphologic approach.

INTRODUCTION
Taxonomic ambiguities are the bane of paleontology
and impede our understanding of evolutionary processes, paleobiological relationships, and paleoceanographic problems. In the taxonomy of Foraminiferida,
one of the dominant microfossil groups in marine sediments, artificial classifications typically are erected on
the basis ofcommon morphological features (Saito and
others, 1981). Whereas the morphological features may
appear to be unifying or differentiating, in actuality
they result from a combination of genotypic and environmental influences. Thus, morphology alone is
sometimes a poor approximation of true genetic associations and taxonomic affinities.
Planktonic foraminifera are greatly influenced by environmental changes in the upper oceanic waters, as
demonstrated by their wide rage of morphologic variability (see Kennett, 1976, for a review). Other independent techniques are therefore needed to clarify taxonomic problems and evaluate subtle differences among
species of planktonic foraminifera. Ideally, a combination of both ecophenotypic and genotypic variation
should be considered in order to construct natural classification schemes and decipher evolutionary trends in
the fossil record.
A major problem in achieving this goal is the lack
of basic genetic information needed to evaluate genotypic features in fossil foraminifera. In living organisms a genetic signature can be isolated from morphologic variation through the use of molecular
information, such as that found in DNA or proteins.
Proteins in general reflect the genetic make-up of an

METHODS
PROTEINBIOGEOCHEMISTRY
To determine species specificity of shell matrix proteins, monospecific samples of Recent planktonic foraminifera were analyzed from the top 2 cm of three
low-latitude cores from the western Pacific, Atlantic,

Department of Geology, University of South Florida, Tampa,

FL 33620.
2 Marine Science Program and Department of Geological Sciences,
University of South Carolina, Columbia, SC 29208.
I

159

160

ROBBINS AND HEALY-WILLIAMS

TABLE1. Locations of cores sampled in this study.

core x

Location

Western Pacific

*ERDC Box 92
ERDC Box I12

Atlantic

*P7008- 10-2,4
V22- 190

Caribbean
Eastern Pacific

*P6304-4-4
P6702- 1-2
P6702-2-4

Tongue of the Ocean,

Bahamas

BX 1
BX8

Cariaco Trench

P6603-3-4

Indian Ocean

V 19-200
V 19-20 1

VI 9-204

Sulu Sea

Circe 18PG
RC14-78
RCI 2-357

Latitude/
longtude

Depth

2"13'S
156'59'E
1'37's
159"14'E
12"59'N
44"20'W
692"
2 I"16'W
15"27'N
70'4 3'W
590"
103"oO'W
2'29"
103"oO'W
25"09'N
77"49'W
2592"
76"54'W
10'48"
64"47'W
4'1 3's
41"33'E
5"20'S
40'26'E
lo"14'S
43'49'E
7"65'N
1 19"oO'E
8'00"
1 18"oO'E
9"oO'N
12095'E

1,598

(m)

2,168

the eluent was monitored at 220 nm for proteins and

peptides. Individual protein peaks collected from this
procedure were first hydrolyzed, and the amino acids
were then monitored by UV absorption at 254 nm after
phenylisothiocyanate (PITC) precolumn derivatization (Robbins, 1987). Reproducibility of area response
of the chromatographs averages 1.5%.

3.6 17

QUANTITATIVE
MORPHOLQGY
4,136
3,159
3,354
1,888

571
2,692

2,000
1,643
2,049

* Refers to cores which were used in the biochemical part of this

study. All cores were used for the image analysis part of this study.
and Caribbean (Table 1). Age estimates for the coretop samples range from 2,000 to 4,000 yr. BP based
on sediment accumulation rates determined by biostratigraphy and oxygen isotope stratigraphy (Emiliani,
1972; Berger and others, 1978; Droxler, 1984; Robbins, unpublished data). Four planktonic foraminiferal
genera were studied: (1) Globigerinoides ruber (&Orbigny) and Globigerinoides sacculifer (Brady), (2) Pulleniatina obliquiloculata (Parker and Jones), (3) Globorotalia menardii (Parker, Jones, and Bandy) and
Globorotalia tumida (Brady), and (4) Orbulina universa
(d'orbigny). Approximately 0.3-0.5 grams of individual species (several thousand monospecific specimens)
were isolated from the >250 pm fraction with a vacuum picker. These specimens were crushed, ultrasonically cleaned in distilled water, and vacuum desiccated. Details of the biochemical methods are presented
elsewhere (Robbins, 1987; Robbins and Brew, 1990).
Dried test material was decalcified in cold 50% formic
acid and centrifuged. The supernatent was exhaustively dialyzed against distilled water in a membrane
with an upper molecular weight limit of 6,000-8,000
daltons. The retained protein material was freeze dried
and separated on the basis of hydrophobicity (charge)
by reverse phase high performance liquid chromatography (RPHPLC) (Robbins, 1987). The absorbance of

To quantify the morphological variability of the foraminifera, approximately 1,200 specimens of each
species were picked from the top 2 cm of 16 widely
spaced tropical cores (Table 1). Specimens were
mounted on standard micropaleontology slides in a
predetermined standard orientation. The two dimensional outline of each specimen was captured via video
digitizer, and the outline coordinates were subjected
to closed form Fourier series analysis (Ehrlich and
Weinberg, 1970), as discussed in Healy-Williams and
Williams (1 98 1). Twenty-four harmonics or shape
components were computed for each digitized specimen. We used EXTENDED CABFAC and EXTENDED QMODEL vector analysis (Full and others, 198 1)
(ECEQ) to determine the morphotypic composition of
the assemblages of each species. Here, we will only
present the morphologic results for the morphotypes
of the species Globigerinoides ruber. The outlines of
all specimens were digitized in a ventral orientation.
All specimens were selected from the 150-250 pm size
fraction to avoid ontogenetic biases.
STABLEISOTOPIC
ANALYSIS
An average of five specimens of each morphotype
of G. ruber was picked from the sediment samples of
Box 1, P6304-4, and ERDC 92 (Table l), and analyzed
for stable oxygen and carbon isotopic composition with
a VG SIRA 24 isotope ratio mass spectrometer. The
monospecific specimens were picked from the 150250 pm fraction to minimize isotope effects due to size
(Curry and Matthews, 1981). White and pink G. ruber
were isotopically analyzed separately. The morphotypes were removed from the samples based on the
ECEQ determined morphotypes, Le., specimens were
removed according to their harmonic 2 amplitude, a
measure of test elongation (Healy-Williams and Williams, 198l). The extraction procedures used in the
isotopic analysis are discussed in detail in Williams
and others (1 988b). The isotopic results presented here
are relative to PDB and have a precision of O.lo/Oo.
RESULTS AND DISCUSSION

PROTEINBIOGEOCHEMISTRY
The important first step in developing a procedure
to ascertain a genetic signature from test material demonstrated that high molecular weight proteins and
polypeptides could be isolated and partially characterized from the fossil organic shell matrices of single
species of planktonic foraminifera (Robbins, 1987;
Robbins and Brew, 1990;Robbins and Donachy, 1990).

161

BIOCHEMICAL, GEOCHEMICAL, AND MORPHOLOGICAL CRITERIA IN FORAMINIFERA

0 G. ruber ( p )
0 0.universa

AMINO ACID COMPOSITION OF FP8

G. sacculifer

2o

P.obliquiloculata
G.tumida
G. menardii

Asx

Glx

Ser
Amino Acids

GlY

Ala

FIGURE1 . Histograms of relative abundances of 5 of the 17 amino acids, expressed as percent of total, found in the FP8 protein from six
species of planktonic foraminifera: Globigerinoides ruber. Orbulina universa, Globigerinoides sacculife, Pulleniatina obliquiloculata, Globorotalia tumida, and Globorotalia menardii. The abbreviations used for amino acids are as follows: Asx = aspartic acid and asparagine; Glx =
glutamic acid and glutamine; Ser = serine; Gly = glycine; Ala = alanine.

The RPHPLC separation of proteins shows that proteins from the six species of core-top planktonic foraminifera have comparable chromatographic profiles.
This similarity indicates that the shell organic matrix
from different species is composed of many of the same
proteins. The amino acid composition of equivalent
chromatographic peaks, thought to represent single
proteins, confirms that the protein composition is indeed similar from species to species, suggesting that
some of the proteins are homologous, although some
amino acid compositional differences were also demonstrated (Robbins, 1987).
The amino acid composition of some of the more
hydrophobic proteins resembles fibrous structural proteins, having significant quantities of glycine, serine,
and alanine (Robbins, 1987; Robbins and Brew, 1990),
which are similar to other structural proteins of Recent
benthic foraminifera (Weiner and Erez, 1984)and mollusks (Wheeler and others, 1987). These results suggest
that structural proteins are responsible for the morphological differences between species and perhaps determine the morphotype of species. These structural
proteins may also be less prone to degradation than
some other types of proteins because of their composition and fibrous structure (Robbins, 1987).
One foraminiferal protein fraction, designated FP8,
was found to be common to all species examined (Rob-

bins, 1987). The compositions of five major amino

acids of polypeptide FP8 from the six species of planktonic foraminifera are shown in Figure 1. This protein
is dominated by the amino acids, aspartic acid asparagine (Asx),glutamic acid + glutamine (Glx), serine
(Ser), glycine (Gly), and alanine (Ala). On the average,
the percentage of Asx in spinose species (10.5% f 1.2)
is always higher than non-spinose species (4.6% f 1.O)
(Fig. I). The percentages of Glx in G. sacculifer (spinose) and P. obliquiloculata (non-spinose) are significantly higher than in the other four species (Fig. l),
but do not distinguish spinose from non-spinose species as in the case of Asx. No significant interspecific
or intergeneric differences were observed in the amino
acids serine and alanine (Fig. I), although the nonspinose group has slightly more glycine ( 1 7.3% f 1.4)
than the spinose group (14.5% f 0.9).
We applied the statistical method of Cornish-Bowden ( 1 983) using 13amino acids to determine if specific
differences exist in the FP8 and to quantify the degree
of relatedness of this protein. Although there are distinct numerical cut-offs for classes of relationships (<42
proteins are strongly related, >93 the proteins are not
related, etc.), this technique can also quantify the degree of relatedness within a category. The results show
that, as expected, the species we analyzed are related
to each other in varying degrees (Table 2). In general,

I62

ROBBINS AND HEALY-WILLIAMS

TABLE
2. Relatedness of FP8 among 6 species of planktonic foraminifera based on statistical method of Cornish-Bowden (1983) as calculated
using mole percent of 13 amino acids and a conservative estimate of molecular weight of 10,000 daltons.
G.

G. ruber
G. sacculifer

0.universa
Gt. tumidu
Gt. rnenordii
P . obliquiloculnta

Nhpr

0
19.6
8.8
57.3
49.3
56.0

G. s'l<mlf<r

0.un;ueno

GI. mwda

GI. menmdii

P. abliquiloculoro

19.6
0
31.7
54.3
49.3
48.7

8.8
31.7

57.3
54.3
47.1
0
27.4
69.5

49.3
50.7
36.4
27.4
0
44.9

56.0
48.7
49. I
69.5
44.9
0

47.1
36.4
49. I

x < 42 proteins strongly related

93 > x > 42 weaker indication of relatedness
x > 93 proteins not related

a biochemical basis exists for the distinction of genera

based on spinose vs. non-spinose morphology and species distinction based on overall amino acid composition of FP8. The only exception to this is the relatedness of Orbulina universa (spinose) to Globorotalia
menardii (non-spinose). The nominal confidence level
of this "strong" test is approximately 92.5% (CornishBowden, 1983) and could indicate a statistical or an
inherent analytical error. For instance, the 0. universa
results indicated anomalously high methionine which
could have affected the outcome of the Cornish-Bowden statistics. However, on a relative scale, the relatedness of 0. universa and G. menardii (36.4) is not as
strong as that between 0. universa and G. sacculifer
(3 1.7). A cluster analysis using all of the amino acids
in FP8 demonstrated a phylogenetic relationship similar to that presented by Kennett and Srinivasan (1983)
(Fig. 2). More specific phylogenetic conclusions from
the molecular data require sequencing of the protein
or may be obtained through other molecular or immunological approaches (Robbins and others, 1990).
However, the amino acid compositional variability that
FP8 displays from species to species (Fig. 1; Table 2)
suggests it has isologous variants that exhibit speciesspecific differences and that can provide additional
phylogenetic information to the micropaleontologist.
Species-specific aspartic acid percentages from bulk
amino acid analyses of spinose and non-spinose planktonic foraminifera and of benthic foraminifera were
previously described (King and Hare, 1972; Haugen
and others, 1989). Bulk analysis, however, is actually
an average of the heterogeneous collection of proteins
within the shell because the organic matrix was found

..

~~

~~

FIGURE2. Dendrogram of results of hierarchical cluster analysis

performed on the amino acid composition of FF'8 from six species
of planktonic foraminifera.

to contain up to seven proteins (Robbins, 1987; Robbins and Brew, 1990). Also, significant amounts of free
amino acids (especially acidic ones, such as aspartic
acid) and small humic acids with hydrolyzable amino
acids are adsorbed onto the test as well (Jackson and
Bishoff, 1971; Mitterer, 1972; Carter and Mitterer,
1978; Robbins and Brew, 1990). These free amino
acids could lead to false taxonomic classification. This
is particularly a cause for concern with aspartic acid,
which has been used for taxonomic distinctions (King
and Hare, 1972; Haugen and others, 1989). Our approach of analyzing the amino acid composition of
specific proteins uses a combination of dialysis, which
eliminates free amino acids, and analysis of high molecular weight protein components. Such techniques
virtually eliminate the possibility of analyzing contaminating free amino acids. Therefore, the results presented here represent a more accurate analysis of the
amino acid composition of a single protein.
The interspecific compositional differences shown
here for FP8 (such as differences in Asx) (Fig. 1) are
not likely dissolution artifacts. For example, the tests
of spinose species are more soluble to bottom waters
that non-spinose species (Berger, 1970), yet the spinose
species have higher percentages of this relatively unstable amino acid (Hoering, 1980). In fact, the compositions are unlikely to be influenced by other environmental factors such as food source differences or
even secondary calcification, since the structural proteins, such as FP8, are genetically predetermined.
Therefore, since the taxonomically useful aspartic acid
and glutamic acid (and possible other important amino
acid) signals are retained, individual proteins in fossil
foraminifera may provide more phylogenetic information (King and Hare, 1972; King, 1980; Robbins,
1987) than that previously obtained using bulk amino
acid analyses.
DISTINGUISHING
MORPHOTYPES
OF
G. RUBER USING
MORPHOLOGY,
STABLE
ISOTOPESAND BIOCHEMISTRY
Both environmental and genetic influences are inherent in the phenotypic expression of foraminifera.
As a consequence, there is considerable difficulty in
distinguishing between intraspecific morphologic variation that is purely ecophenotypic, and that which re-

163

BIOCHEMICAL, GEOCHEMICAL, AND MORPHOLOGICAL CRITERIA IN FORAMINIFERA

A.
IOW

<

Elongation

high

A.

W.PACIFIC G. RUBER

B.

MORPHOTYPES

TOTO G. RUEER
MORPHOTYPES

eo

' 0 ~
10'

-2.75

- -2.25
- 2.0
-1.75
-1.5

c.

Z
0
Y

!-:
I 20

1.25

1.5

"I

1.75

Del

GI3

2.25

2.5

2.75

0 G ruber-'normal' Ipl

0 G ruber-G

elongatus
G menardii

(wl

- I S?

"10.
21

16

20,

12

8
4

0
As%

Glx

Met

Thr

Val

Amino Acids

FIGURE
3. Diagrams showing morphological, isotopic, and biochemical data for morphotypes of G. ruber: A. The six morphotypes
of G. ruber as quantified fmm 16 core tops from the tropics. Morphotypes were distinguished on the basis of chamber elongation
(harmonic 2) as determined by EXTENDED CABFAC and EXTENDED QMODEL vector analysis. Increasing elongation between
the ultimate and penultimate chambers is shown from left to right.
B. Graph of carbon versus oxygen isotope values for six morphotypes
from TOTO Box 1 shown in Figure 3A. As shown, two groups, A
and B, are delineated, and show wide variation in values between
the groups. C. Histograms showing relative percentages of 5 of the
total 17 amino acids comprising FPS protein from a morphotype
representative of isotopic groups A and B. Globigerinoides ruber
"normal" from group B and C. elongatus from group A were chosen
on the hasis of shape difference (harmonic 2), calor differences (p =
pink versus w = white), andisotopic separation. Sampleswerepicked
from Atlantic core top P7008. Also, included for comparison are
compositional data from a morphologically unrelated species, Globorolnlia menardii. The abbreviations used for amino acids are as
in Figure 1 and Thr = threonine and Met = methionine.

sults from genetic differentiation either in concert with,

or caused by, environmental differences. Based on the
results ofthe protein analysis, we undertook a detailed
morphological, stable isotopic, and biochemical study
of the planktonic foraminiferal species Globigerinoides
ruber in order to determine the genetic relatedness of
the morphologic variants of this species. Globigerinoides ruber exhibits considerable morphological variation, and it remains unresolved if the morphotypes
represent separate species or environmental variants

Amnlitude

Amplitude

FIGURE4. Histograms of proportions of Globigerinoides ruber

end members in a sample. A. West Pacific ERDC Box 92 shows
high proportion of EM I and EM 6 individuals. B. Histograms of
Tongue of the Ocean VOTO) sample shows that it is dominated by
EM 1, 2, and 6 individuals.

(Parker, 1962; Hecht and Savin, 1972; Hecht, 1974;

Emiliani, 1974). Although a number of the morphotypes of G. ruber have been recognized as genetically
distinct species, for example, G. elongatus (d'orbigny,
1826), G. pyrumidulis (van den Broeck, 1876), and G.
cyclostomus (Galloway and Wissler, 1927), the suggestion that these morphotypes are phenotypic variants (Kennett and Srinivasan, 1983) is at least partially
supported by ecological studies (B6, 1977).
The morphologic variability of 1,200 specimens of
G. ruber that are 150-250 Fm in diameter, was examined via closed-form Fourier in a previous study of
the 16 core tops listed in Table 1 (Robbins, 1987). A
maximum entropy analysis (Full and others, 1984) of
the harmonic amplitude spectra indicated that harmonic 2, a measure of shell elongation, contained the
maximum amount of morphologic variability. An EXTENDED CABFAC EXTENDED QMODEL vector
analysis of the frequency distributions of harmonic 2
indicated that six morphotypes account for more than

164

ROBBINS AND HEALY-WILLIAMS

PLATE I
Morphotypes of Globigerinoides ruber based on the results of closed form Fourier series analysis, EM 1 through EM 6, from left to right,
in order of increasing elongation (harmonic 2). Scale bar = 50 pm.

95% of the variance in the data array. These six morphotypes, called end members (EM 1 through EM 6),
represent increasing degrees of elongation that are
schematically depicted in Figure 3A and Plate 1. Not
all end members are present in all samples examined.
For example, the western Pacific is dominated by a
bimodal distribution of end members 6 and 1, but has
no specimens typical of end member 4 (Fig. 4A). Tongue
of the Ocean, Bahamas (TOTO) was found to be dominated by end members 1, 2, and 6 (Fig. 4B). We interpret this to indicate geographic variability in the
dispersal of these forms.
Specimens of G. ruber morphotypes from Box 1,
TOTO Bahamas, were subsequently analyzed for their
stable isotopic compositions (Fig. 3B). From the six
identified morphotypes (EM 1 4 ) , two general groups
of end member associations can be distinguished on
the basis of their oxygen and carbon isotopic composition. Group A contains the less elongate morphotypes
EM 1-3, which on the average are depleted in 6I8O
and 6I3C by approximately - 1 . 4 7 ~and - 1 . 2 5 7 ~respectively, compared to the more elongate morphotypes in Group B (EM 4-6). Similar isotopic/morphologic trends for this species were seen in the western
Pacific, Caribbean, and other Atlantic cores (Robbins,
1987). Erez (1 979) determined an oxygen isotopic difference which averaged 0.5700between G. ruber and G.
elongatus from plankton tows. The estimated oxygen
isotopic value for equilibrium calcite at the TOTO site
is - 1.47~,close to the value of EM 5 G. ruber. The
oxygen isotopic difference found between extremes for

the two groups (1.57~)is the typical 6I8O difference

found between glacial-interglacial samples (Lynts and
Judd, 1971). It is unlikely that the range of values
observed in the G. ruber morphotypes is due to bioturbation since the Tongue of the Ocean is a region of
relatively high sedimentation rate (Lynts and Judd,
197 l), which significantly reduces the effect of vertical
mixing. Average sedimentation rate for Box 1 is approximately 7-1 5 cm/yr3 (Droxler, 1984). The range
in isotopic values could be the result of seasonal temperature changes at the site. Globigerinoides ruber is
known to have a seasonal component in both its oxygen isotopic composition (Williams and others, 1979,
1981) and morphology (Spero and others, 1987). A
seasonality effect does not eliminate the possible genetic component to the differentiation of the morphotypes of G. ruber. These morphotypes might indicate
that these organisms are utilizing particular niches. We
therefore suggest that the 6I8O differences between
morphotypes groups A and B are neither due to bioturbation nor purely ecological factors.
Unlike the 6I8O of foraminifera, 6I3C is not a function of temperature but rather a function of how each
species incorporates the bicarbonate ion during calcification. Such 6I3Cdifferences as those seen in G. ruber
morphotypes have often been attributed to the catchall term vital effects that may actually reflect a number of phenomena, including genetic variation (Weiner, 1975; Williams and others, 1977; Erez, 1978).
To examine possible biochemicalhiological differentiation in G. ruber as related to the isotopic and

BIOCHEMICAL, GEOCHEMICAL, AND MORPHOLOGICAL CRITERIA IN FORAMINIFERA

morphological differences of groups A and B, we determined the amino acid compositions of the protein
FP8 from one morphotype in each of the two isotopic
groups (EM 1 from Group A and EM 5 from Group
B) (Fig. 3C). For this analysis we used Atlantic core
P7008, which contained these two morphotypes in
abundance and had similar isotopic/morphologic
trends. These two forms were chosen because they are
morphologically distinct due to their degree of elongation (Plate 1; Fig. 3A). Figure 3C shows a partial
amino acid composition of the individual FP8 proteins
in the two G. ruber morphotypes. These preliminary
data show that only a few differences in the amino acid
composition of FP8 exist between the two morphotypes of G. ruber. A small number of mutations (amino
acids that have replaced each other) may be indicative
of less genetic divergence of the proteins (and therefore
of the species) as compared to larger differences seen
in FP8 between species and genera. Also notable is the
fact that the amino acids, which show variation between G. ruber morphotypes, are some of the more
conserved amino acids. They are relatively immutable
during evolution as compared to the highly mutable
amino acid, aspartic acid (Creighton, 1983). In structural proteins of taxa from sponges to humans, small
differences in amino acid composition are unique and
can be used taxonomically (Balazs, 1970; Fietzek and
Kuhn, 1976; Furthmayr and Timple, 1976; Lowenstein,
1980). While more data are needed to investigate the
biochemical relationships between EM1 and EM5, the
differences in amino acid composition of the FP8 protein found in the two morphotypes are consistent with
our interpretation of the morphologic and stable isotopic results indicating that there are two distinct groups
of G. ruber. In a separate line of investigation, Deuser
and Ross (1989) have also suggested a non-environmental distinction between the two varieties. The combined biochemical, morphological, and geochemical
results support the distinction of G. ruber morphotypes
in terms of genetic rather than environmental causes.
LIMITATIONS
OF THE PRESENT
BIOCHEMICAL
RESULTS
In order to fully understand the relationship between
the planktonic foraminiferal morphotypes and differences in protein amino acid composition, sequence
data are required. Sequencedata will allow comparison
of the amino acid sequence from one species to another, which will provide information concerning degree of relatedness and give an indication of the time
of branching events in the phylogenetic history of the
lineage. One problem that presents itself is the amount
of material needed to sequence proteins. In order to
fully sequence the FP8 protein from the two morphotypes of G. ruber examined in this study, hundreds of
thousands of specimens (approximately 5-1 0 grams)
are needed. While obtaining large amounts of sediment
may not be an issue, the limiting factor is the time
involved in the picking of a monospecific sample. Another obstacle is that sequencing the shell matrix pro-

165

teins is not a straightforward procedure: at least one

of the proteins is blocked to Edman degradation (Robbins and Donachy, 1991) and therefore limits the
amount of protein that can actually be sequenced.
However, it appears likely that partial sequences can
be obtained (Robbins and Donachy, 199 1). These partial sequences may still allow phylogenetic relationships to be explored because of unique domains in the
protein structure. In the next few years it can be anticipated that technological limitations will be reduced.
If one compares the vast numbers of specimens needed
by Emiliani (1 954) for his original stable isotope work
to the few specimens required by present day mass
spectrometers, one can look toward technological improvements to eventually eliminate the problem of
large sample size. In addition, there may be more efficient ways to determine a genetic signal from living
foraminifera through the analysis and sequence of DNA
or through the use of monoclonal antibodies (Robbins
and others, 1991).
IMPLICATIONSOF THISSTUDY
The goal of this preliminary research is to provide
the foundation from which we can eventually infer
evolutionary relationships. Since slight modifications
of morphology may be due to either environmental or
to genetic influences, morphologicdata alone are sometimes not sufficient to make taxonomic distinctions in
micropaleontology. As a first step, we show here that
the protein FP8, present in different species of core top
planktonic foraminifera, shows compositional differences reflecting species differences. We have further
shown that there are stable isotopic differences between
the morphotypes of G. ruber and that a portion of these
differences may be the result of genetic variation between morphotypes rather than ecologic variation.
Further evaluation of the small compositional differences between morphotypes is currently in progress.
The integration ofa biochemical signature with morphologic and isotopic data may aid in the refinement
of taxonomic lineages, thereby improving biostratigraphy. Successful sequencing of the proteins will enable
us to determine the substitutions, deletions, and insertions of amino acids that have occurred within the
polypeptide chain. During speciation, new protein
structures gradually emerge from old ones as a direct
consequence of changes in genetic material along different lines of descent (Doolittle, 1979). Until more is
known about foraminiferal DNA (the genotype), protein data, a close approximation of the genotype, will
allow us to compare proteins within and between species and explore evolutionary histories of species. In
addition, evolutionarily conservative proteins can be
recognized by comparing the diverged sequences and
the less conservative proteins can be used by taxonomists. Phylogenetic trees based on a combination of
molecular, morphologic, and isotopic evidence might
be created much in the same way that micropaleontologists now construct them based solely on morphology. The addition of a proxy genetic component

166

ROBBINS AND HEALY-WILLIAMS

from the uniquely complete depositional sequences

provided by deep-sea sediments could lead to a better
understanding of organic evolution and how phenotypic variations relate to ecologic domains, to evolution of lineages, and to speciation.
ACKNOWLEDGMENTS
Acknowledgment is made to the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this research
(LLR) and National Science Foundation grants
88 17343 (LWR) and OCE 86 14073 (NHW). Also acknowledged are the Gulf Coast Association of Geological Societies and the Bader Fund. We are also grateful to the following people who greatly aided in this
research: K. Brew, R. Ehrlich, D. Williams, C. Emiliani, and L. Peterson. We also appreciate the efforts
ofJohanna Resig, Bjorn Malmgren, and an anonymous
reviewer in reviewing this manuscript.
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Received I 9 January I990

Accepted 23 May I990