Download Phylogenetic relationships among the species of genus Bulimina

FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1
Phylogenetic relationships among the species of genus Bulimina
(Foraminifera) inferred from ribosomal DNA sequences
Hiroshi Kitazato1, Masatoshi Tazume2 and Masashi Tsuchiya3
Research Program for Paleoenvironment, Institute for Frontier Research on Earth Evolution (IFREE)
Department of Life and Earth Sciences, Shizuoka University
3
Marine Biosystems Research Center, Chiba University
1
2
Introduction
Materials and methods
Because their distribution patterns are related to environmental parameters such as temperature and salinity, benthic
foraminifera have long been used as excellent proxies for
reconstructing paleoceanographic parameters. Among
foraminiferal genera, species of the genus Bulimina distribute
in a wide range of oceanic environments from sublittoral to
bathyal depths and have clear habitat preferences. Thus,
Bulimina species are thought to be useful foraminiferal taxa
for paleoceanographic application. However, there has been
considerable discussion concerning the definition of species
that are mainly defined only on the basis of morphological test
characteristics.
One major discussion has focused on whether or not the
morphology-based systematics of the genus Bulimina is reliable. Within this genus, two groups have been recognised, one
with costate and the other with non-costate test surface ornamentation. Verhallen (1986) considered that costate surface
ornamentation, together with the presence of an internal tooth
plate, is a diagnostic character for discriminating one group
from the other. He suggested that these morphological characteristics are very important taxonomically, and might even
represent family-level differences.
On the other hand, gradual morphological changes also
exist among several species of Bulimina. Both Bulimina marginata and Bulimina aculeata were described on the basis of
modern specimens collected in the Adriatic Sea by d’Orbigny
(1826). He described both forms as distinct species on the
basis of chamber angularity, the arrangement and length of the
spines, and test dimensions. Hoeglund (1947) made precise
morphological analyses of both species and found that morphological characterics changed gradually from one species to
the other. He concluded that they were not separate species but
morphological variants of the same species. However, both
Collins (1989) and Burgess and Schnitker (1990) concluded
that B. marginata could be distinguished from B. aculeata
based on morphological characteristics.
These conflicting views may arise due to a lack of knowledge on the biological significance of the morphologies. Biological approaches, such as ecological analysis and molecular
analysis, may help to solve the problem. In particular, molecular phylogenetic analysis of Bulimina species provide an adequate approach to evaluate the significance of morphological
characters for distinguishing taxa. The aim of this study is to
analyze molecular phylogenetic relationships among the
species of genus Bulimina using nucleotide sequences, and to
discuss the relations between molecular- and morphologybased classifications.
Samples
Eight morphospecies were used for DNA study. Living
individuals for study were collected at different localities from
sublittoral to bathyal depths around the Japanese Islands (Fig.
1). Sublittoral sediments were collected from Kominato Fishing Port at Uchiura Cove of Chiba Prefecture, Shiogama Fisherman’s Warf at Matsushima Bay of Miyagi Prefecture, and
Shimoda Bay of Shizuoka Prefecture. Bathyal sediments were
collected from Stns. SB, G and EO in Sagami Bay. The samples with living Bulimina species were maintained in aerated
buckets in the laboratory at about 20°C for shallow water
species, and 2.5°C for deep-sea species, until DNA analyses
were conducted.
Molecular phylogenetic analysis
The specimens for DNA analysis were picked out from the
cultured sediments. Individuals with cytoplasm inside the test,
and/or extruded pseudopodia from the aperture, were judged
as alive. To remove any associated microorganisms, individual
foraminifera were cultured without food at 18°C in a petri dish
filled with 0.2µm filtered and sterilized seawater (35‰). After
2 or 3 days, a period by which most ingested food material
should have been digested, individuals were dried at 30°C for
30min. (Holzmann and Pawlowski, 1996). Prior to extracting
DNA, dried specimens were photographed using a low vacuum SEM (JEOL) in order to compare DNA sequence data
with morphological information.
DNA extraction
Total DNA was extracted from a single individual. A single
dried specimen was transferred into a 1.5ml microfuge tube
with extraction buffer containing 50µl of 1x DOC (Pawlowski
et al., 1994). The specimen was crushed with a siliconized and
tip-closed Pasteur pipette, and then incubated at 60°C for 1
hour. Insoluble materials were separated by centrifugation.
Supernatant was collected and used for PCR amplifications.
Amplification and purification
A DNA fragment of about 1,000 base pairs (bp), situated at
the 3’ terminal region of the SSU rDNA, was amplified by
polymerase chain reaction (PCR) using synthesized primer
pairs. Primer pairs of s14f1 (5’-AAGGGCACCACAAGAACGC) and sB (5’-TGATCCTTCTGCAGGTTCACC233
FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1
morphospecies ranged from 0.0 to 5.2% (Table 1). As in the
case of sequence divergence within an individual, most
species show a divergence of less than 1.0%. An exception is
Bulimina kochiensis in which sequence divergence within a
population ranged from 1.1% to 4.8%. There is also a 3.0%
sequence divergence within B. marginata forma denudata.
Among species of the genus Bulimina, sequence divergence
reached a maximum of 35.0%. The divergence between B.
aculeata and B. pagoda was smaller (0.1-0.6%) than between
other species. A low degree of divergence was also observed
within the group B. aculeata, B. marginata, B. elongata and B.
pagoda. However, the divergence between B. kochiensis and
B. striata was significant, with a value of 34.5-35.0%. Apart
from these extreme values, divergence clustered around 15%.
Sequence divergence between B. kochiensis and other morphospecies was around this value; for instance, between B.
kochiensis and B. aculeata it ranged from 15.5% to 16.3%.
Phylogenetic relationships among species of Bulimina indicate that the genus consists of four different genetic clusters
(Fig. 3). The sequence divergences between these clusters are
large. Phylogenetic relationships of the four Bulimina clusters
with other genera of foraminifera show that three of the four
groups are scattered within the phylogenetic tree (Tazume et
al., in prep.). Each cluster exhibits characteristic morphological features, such as pore size, pore distribution, density distribution of pores, aperture shapes and test ornamentation. Other
characters, such as test chemical composition, overall outline
of tests, and the shape of the loop-like aperture, are common
to all species of Bulimina. This means that morphological convergence has occurred in the genus Bulimina.
The main reason for the large genetic difference between
the species clusters was the existence of either insertions or
deletions among genetic fragments at the SSU section. This
phenomenon, insertion or deletion, is very common in
prokaryotic genes but rarely found in eukaryotic metazoans.
Further investigations are needed to determine why genetic
insertion or deletion often takes place in certain foraminiferal
genera.
Five morphospecies, Bulimina aculeata, B. pagoda, B. marginata forma marginata, B. marginata forma denudata, and B.
elongata, are genetically very close within the Bulimina
group. They all belong to a single cluster in a phylogenetic
tree (Fig. 3). Four subcluster units could be recognized within
the main cluster (Fig. 4). Some morphotypes, such as B. elongata, B. marginata forma denudata and B. marginata forma
marginata, corresponded to a single subcluster. Both Bulimina
aculeata and B. pagoda, however, belong to different subclusters with B. elongata and B. marginata. Thus, morphological
divergence may occur among closely related species of genus
Bulimina.
TAC) were used for SSU amplification. These primers were
designed by Pawlowski et al. (1994; 1996).
PCR amplifications were performed in initial denaturation
for 2min. at 94°C: 1 cycle, denaturation for 30sec. at 94°C,
annealing for 30sec. at 55°C, and extension for 90sec. at 70°C:
40 cycles, annealing for 2min. at 55°C, final extension for
7min. at 72°C: 1 cycle. The reactions were performed with
5.0µl of 10x reaction buffer, 5.0µl of dNTP mixture (contain
2.5mM each of dATP, dCTP, dGTP, and dTTP), 2.5µl each of
forward and reverse primers (10pM), and 0.5µl of Taq polymerase (2.5U), for 50µl reactions. The length of the fragment
was confirmed by agarose gel electrophoresis. PCR products
were purified with High Pure PCR Product Purification Kit
(Roche). Purified products were stored at –20°C.
Cloning and sequencing
The purified products were ligated in the pGEM-T vector
System (Promega) and cloned into XL-2 blue ultracompetent
cells (Stratagene). Plasmids were isolated from cell cultures by
the mini-preparation methods (Sambrook et al., 1989). The
sequencing reaction on the DNA fragments was carried out the
using Thermo Sequenase pre-mix cycle sequencing kit (Amarsham Pharmacia) with Texas Red labeled primers. The samples
were sequenced using an Hitachi SQ-5500 DNA sequencer.
Phylogenetic analysis
The DNA sequences were aligned using Clustal W
(Thompson et al., 1994) and corrected manually. Estimation of
sequence divergence was calculated by the two-parameter
method of correction of multiple substitutions at a site (Kimura, 1980) (transition/transversion ratio=2.0). The unrooted
trees were constructed by neighbor joining (NJ) (Saitou and
Nei, 1987) and were performed using Clustal W. Bootstrap
was with 1,000 replicates for NJ.
Results and discussions
The amplified SSU rDNA fragment of species of the genus
Bulimina corresponds to the 3’ terminal region of the SSU
rDNA of Mus musculus (X00686), starting at position 1,208
and ending at position 1,866. The length of the SSU rDNA
fragment varies from 981 to 1,232bp between different morphospecies of Bulimina. The small subunit rDNA fragment
length of most morphospecies is 1,000 bp on average,
although Bulimina striata has longer sequences (1,232bp). In
contrast, fragment length (981bp) is rather short in Bulimina
subornata. The diagram is illustrating alignment of two distinct species, B. striata and B. subornata (Fig. 2) indicateds
that either insertion in B. striata or deletion in B. subornata
may occur in any part of the DNA sequences. Results from
alignment of all species indicate that the sequenced region
consisted of both conserved and variable regions (Fig. 2).
Variable regions occupied about 650bp of the total 1,338
aligned sequences. The differences in the length of DNA fragments for each species originated in the variable region. G+C
contents are stable within the genus Bulimina, ranging from
42.8% to 46.9%.
The sequence divergence within a population of a single
Summary
DNA analyses were carried out on SSU rDNA for eight
morphospecies of the genus Bulimina. The genus consists of
four genetic clusters. Large sequence divergences were detected among Bulimina species. The differences may exceed those
at the family level, despite the fact that these morphospecies
belong to one genus. Morphological convergence may also
have taken place within the genus Bulimina. Large sequence
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differences are mainly due to either the insertion or deletion of
gene fragments within the variable region.
Five out of the eight morphospecies belong to one genetic
cluster. However, each morphospecies constitutes different
subcluster within the main cluster. Morphological divergence
has taken place in these morphospecies. Thus, both morphological convergence and divergence may have occurred within
one genus of foraminifera.
Further research is needed to clarify the genetic features of
foraminifera using molecular, morphological and ecological
information. This kind of approach may reveal why benthic
foraminifera are finely attuned to specific environmental factors.
Acknowledgments. The authors heartily thank the staff of Misaki
Marine Biological Station and R/V Tanseimaru of the Ocean Research
Institute, University of Tokyo, Shimoda Marine Research Center of
Tsukuba University, and Marine Biosystems Research Center, Chiba
University for collecting living Bulimina individuals. Dr. Andrew J.
Gooday kindly read the earlier version of the manuscript and gave
helpful comments. This research was partly supported by JSPS
Research Fellowships (no. 8489) to M.T., and Grants-in-Aid from the
Ministry of Education, Science and Culture of Japan (nos. 0645002
and 09554025) to H.K.
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Figure 1. Map showing sampling localities. Eight morphospecies
were used for DNA analysis.
Figure 2. Diagram of aligned sequences of buliminid species within the SSU region. SSU rDNA region consisted of both conservative and variable parts.
Shaded area shows conservative part of SSU region. Either insertion or deletion is shown in the variable section.
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Figure 3. Unrooted phylogenetic tree indicates four major clusters recognized among eight morphospecies of genus Bulimina. SEM photographs are
attached in each morphospecies. Scale bar=100µm.
Figure 4. Phylogenetic relationships among five morphospecies of genus Bulimina based on SSU rDNA sequences. Each morphospecies roughly corresponds with one subcluster. Both B. aculeata and B. pagoda constitute one cluster.
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Table 1. Sequence differences among eight morphospecies of genus Bulimina for SSU rDNA. This table also indicates sequence divergences among individuals in one population.
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