Diversity of Polar Monothalamous Foraminifera

Morphological and Molecular Approach

Anna Sabbatini
Department of Marine Sciences, Polytechnic University of Marche,
Via Brecce Bianche, 60131, Ancona, Italy
Introduction
In many marine areas, Foraminifera are a dominant meiofaunal group in terms of both numerical
abundance and biomass. Monothalamous (single-chambered) species with either agglutinated or
organic-walled shells (allogromiids in the broad sense) are an important component of marine
foraminiferal assemblages, particularly in fine-grained sediments, for example in the deep sea and in
high latitudes regions (Gooday, 2002). Monothalamous Foraminifera also occur in freshwater
environment (Holzmann et al. 2002, 2003) and have even been discovered to live in a damp terrestrial
habitat (Meisterfeld et al. 2001). These delicate Foraminifera often account for 10-20% of individuals
and species in deep-sea samples (Gooday, 2002). In Polar coastal regions, influenced by turbid glacial
melt water, and in some estuaries, allogromiids (including saccamminids) represent an even higher
proportion of live Foraminifera, in some cases >90% (Gooday, 2002).
Despite of their high abundance and ecological importance, the diversity of monothalamous
Foraminifera is very poorly known. Relatively few species have been described and most of these come
from easily accessible, intertidal environments. Deeper sea samples routinely yield ten or more
undescribed species (Gooday et al. 1998). Several new morphotypes of allogromiids have also been
reported from Antarctic coastal waters (Gooday et al. 1996). Several new species of polar and subpolar
monothalamous Foraminifera have been described (Gooday et al. 1995; DeLaca et al. 2002; Wilding
2002) or redescribed (Bowser et al. 2002) during the past few years. Because the morphological
characters are not always sufficient to establish the phylogenetic relationships among allogromiids,
many recent species descriptions include the analysis of molecular data (Pawlowski et al. 2002a;
Cedhagen and Pawlowski, 2002; Gooday et al. in press; Gooday and Pawlowski, submitted; Sabbatini
et al., 2004).
Molecular phylogenetic studies challenged the traditional, morphology-based classification of
Foraminifera, prompting a profound revision of higher-level taxonomy and species identification
(Pawlowski 2000, Pawlowski and Holzmann 2002). Phylogenetic analysis of molecular data, based
mainly the small subunit ribosomal RNA and actin gene sequences, revealed that the Foraminifera
include not only testate marine species but also the naked freshwater amoeboid protists (Pawlowski et
al. 1999). Molecular studies also show that there is no clear boundary between allogromiids in the
traditional sense (i.e. organic-walled species) and single-chambered agglutinated Foraminifera,
traditionally considered as astrorhizids, suggesting that all of them form a paraphyletic group of
monothalamous Foraminifera (Pawlowski et al., 2002a, 2003). A particularly high taxonomic diversity
of allogromiid Foraminifera, represented in majority by undescribed species, was revealed by molecular
study of Antarctic Foraminifera (Pawlowski et al. 2002b).
At the beginning of project the main objective was to describe new Antarctic monothalamous
Foraminifera. Many of these Foraminifera were isolated and molecularly characterized in preceding
study (Pawlowski et al. 2002c) but could not be attributed to any known species. During the year there
was possibility to collect other samples but in a different site and environment (Porcupine Abyssal
Plain, NE Atlantic Ocean). The morphological analyses carried out on board on superficial sediment
samples revealed an interesting monothalamous foraminiferal fauna composed especially by specimens
belonging to Komokiacea group. These specimens are really poor known and the most recent studies
are by Schroder et al. (1989), Gooday (1982, 1983), Gooday & Cook (1984), Tendal (1972, 1979),
Tendal & Hessler (1977), Kamenskaya (1990, 1993).
Since there are no sequences available of Komokiacea yet, and several samples of them were available
also from Antarctica to study, new aim was describe and identify a larger number of komokiacean
specimens from these two sites and where possible correlate them. Important goals are 1) molecular
data of Komokiacea group from the Atlantic Ocean in order to reveal them taxonomic diversity and
phylogenetic position within the Foraminifera 2) a comparison of species present in the Antarctic and
NE-Atlantic areas of study 3) morphological description of new species. I focused on the above points
1 and 2.
Komokiacea (Textulariina, Foraminiferida, Protista) are agglutinated Foraminifera with test consisting
of a complex system of fine, branching tubules of even diameter. The test wall is simple, with
argillaceous particles. Stercomata accumulate within the tubules. The group has worldwide distribution,
particularly in the abyssal zone. The greatest relative abundances have been found in abyssal
oligotrophic areas and in hadal trenches. Tendal & Hessler (1977) divided Superfamily Komokiacea
in two families Komokiidae (Tendal & Hessler, 1977) and Bacullelidae (Tendal & Hessler, 1977). First
have test bushy or arborescent constructing widely spaced branching cylindrical tubules and second test
variously shaped, sparsely branched or dense clump. Branching is the basic feature controlling body
form. In most cases branching is dichotomous, although tri- or polychotomy also exists. In the tube wall
two layers can be distinguished: the inner layer is generally very thin and frequently laminated while
the outer part is composed by agglutinated sediment. Komokiacea are devoid of real apertures. The
pseudopodia must penetrate the tube wall through minute pores having possibly temporary character
(Tendal & Hessler, 1977). The Komokiacea vary in according to the amount and type of particles in the
agglutinated layer.
Material and Methods
Specimen collection
The material for this study was collected in the Weddel Sea (Antartica) during R/V Polarstern cruise
ANT-XIX/4 (ANDEEP II, February 28th to April 1st, 2002) (Figure 1).
The other material was collected in the Porcupine Abyssal Plain (NE Atlantic Ocean) in June 2004
(Figure 2). The Foraminifera (not only Komokiacea for a total of 80 samples) for molecular work have
been isolated and stored in guanidine. Sediment samples for morphological studies were sieved on 300
micron mesh and preserved in formalin 10%. All monothalamous soft-walled Foraminifera (organic
and agglutinated) will be sorted by hand, under a stereoscopic microscope, from the floated residue.
Individuals will be placed in glycerol on a cavity slide, described, photographed and classified.
DNA extraction, amplification and sequencing
DNA was extracted from single or several cells using the guanidine method as described in Tkach
& Pawlowski (1999). PCR amplifications were performed in a total volume of 50 :l with an
amplification profile consisting of 40 cycles of 30s at 94/C, 30s at 48/C and 120s at 72/C, followed by
5 min at 72/C for final extension. A fragment of the SSU rRNA gene was amplified by PCR with the
primer pair s14F3 (5ACG CA(AC) GTG TGA AAC TTG) and sB (5 TGA TCC TTC TGC AGG TTC
ACC TAC). When the first PCR was unsuccessful, the PCR products were re-amplified using the nested
primer s14F1 (5' AAG GGC ACC ACA AGA ACG C), with an amplification profile consisting of 25
cycles and 52/C for annealing time. A second series of analyses were carried out using the primer pair
s14F3 (5ACG CA(AC) GTG TGA AAC TTG) and s17 (5CGG TCA CGT TCG TTG C) for the PCR
and the same nested primer (5' AAG GGC ACC ACA AGA ACG C) for the re-amplification. The
amplified PCR products were purified using High Pure PCR Purification Kit (Roche Diagnostics), then
either sequenced directly or ligated into pGEM-T Vector system (Promega) and cloned in XL-2
Ultracompetent Cells (Stratagene). Sequencing reactions were prepared by using ABI-PRISM Big Dye
Terminator Cycle Sequencing Kit and analysed with an ABI 3100 DNA sequencer (Perkin-Elmer), all
according to the manufacturers instructions.
Sequence analysis
Sequences were aligned manually to the large database of foraminiferal sequences, using the
Seaview software of Galtier et al. (1996) and Bioedit program (2004). A fragment of 462 sites was used
for the analysis with sB primer and a fragment of 100 sites was used for analysis with s17 primer.
Phylogenetic analyses were performed with the neighbor joining method (NJ) using Kimura 2 distance
algorithm.
Result Morphological work
Here I present a short taxonomic survey of some of the must abundant larger (>300 micron) rhizopod
species, representing a wide range of taxa, from populations on both the Porcupine Abyssal Plain and
Weddel Sea.
The species and morphotypes from both study sites were divided into taxonomic categories in most
cases genera such as Ipoa spp., Komokia spp., Lana spp., Normanina spp. and Septuma spp. but also
indeterminate groups, e.g. Chain spp.
Chain-like komokiaceans:
These are common in the Porcupine Abyssal Plain and it is possible distinguish 7 morphotypes. The
chains often consist of a branched or occasionally branching chain of small spherical chambers
connected by long, narrow, gently tapering necks. Occasionally globigerinacean tests of varying size
are incorporated within the chamber wall. Other types are organized as a rigid chain of irregularly-
shaped, elongate chambers connected by narrow necks. The chambers are angular; long narrow tubules
of even width may extend from the end of the chamber. The test wall consists of globigerinacean tests
of variable size. Similar morphotypes are distinguished from these forms by the irregularly-shaped
chambers as well as by chambers separated by rigid, short, narrow necks. Other Chain-like forms
consist of a series of spherical/ cylindrical chambers which adjoin directly; the chambers have a bumpy
exterior surface, incorporating globicerinacean tests of varying size incorporated. Occasional long,
single filaments may extend from the surface. Sometimes irregularly shaped chains of chambers are
densely encrusted with globicerinacean tests which obscure the basically organic test wall and make
individual chambers difficult to distinguish. Also in this case occasional short, fine, transparent fibres,
and long, white filaments, extend from the surface. In other species, a branching chain of triangular,
claw or arrow-head shaped chambers joined together at their pointed ends to produce a chain with an
elegant appearance. Finally, a distinctive morphotype comprises pear-shaped chambers joined to form
a branching chain which undergoes characteristic angular changes of direction. The chambers are joined
head-to-toe (Plate 1).
All chain-like morphotypes are quite delicate, they break easily and are typically found in a fragment
state.
Komokia-like group
This group is dominant in samples from Porcupine Abyssal Plain area; some specimens Komoki-like
were found also in the Antarctic study area.
According to Tendal & Hessler (1977), Komokia morphotypes have generally bush like body
consisting of mostly dichotomously branching tubules without anastomoses, the branching points
occurring at any place along the tubules. Ramification increases toward periphery of body. All tubules
are the same diameter (more less), and are nonseptate (Tendal & Hessler, 1977).
In our material the interstices between the tubules are sometimes filled with abundant sediment and the
general body form is elongate, pear or club-shaped body. Long, umbranched, empty filaments of
varying length, frequently extend from the surface. Specimens often differ from classical representatives
of the genus is consisting of only a few tubules. Globigerinacean tests are occasionally attached to
branching tubules (Plate 2).
Genus Lana and Reticulum
Species of the closely-related genera Lana are common in both study areas. Lana species have the form
of a loose mass of branching and anastomosing tubules with no centre of organization or pattern of
growth. The tubules are parallel sided and non-septate (Tendal & Hessler, 1977). In Reticulum the
tubules anastomose more extensively to than in the case of Lana. It is possible to recognize a large
number of morphotypes and classification is based on form of the clump, on the frequency of
anastomoses, and on the degree of filling of interstices with sediments (Gooday & Cook, 1984).
The species of Lana and Reticulum studied vary from irregularly-shaped clump of fine, or very fine,
branching and anastomosing tubules of even diameter to a pear-shaped, tightly packed clump of tubules
of even diameter. There are also morphotypes represented by coherent, spongy, irregularly to
spherically-shaped masses of regularly anastomosing tubules or loose network of branching tubules
(sometimes fine) anastomosing, and not always of even diameter. The interstices of the Lana/Reticulum
tubule system are either devoid of sediment or sediment-filled; the tubules may be transparent and
tipically contain dark masses of stercomata. Globigerinacean tests are often incorporated within the
tubules network or throughout tubule clump (Plate 3).
Ipoa group
Morphotypes belonging to Ipoa group are better represented in the Antarctic environment.
Most specimens are tree-like fragments treelike with a basal stem followed by burst of tightly spaced
multiple branching; diameter of tubules decreases with each branching. Tubules are avoid of septa
(Tendal & Hessler, 1977) (Plate 4).
Normanina group
Also specimens belonging to this group are also more common in the Antarctic although there are some
PAP representatives.
They have a base or center composed of tubules branching in complex, irregular pattern. Tubules radiate
outward from this base and terminate in globular or clublike chambers (Tendal & Hessler, 1977) (Plate
4).
Septuma group
Species of Septuma group in the Antarctic samples are represented by specimens with bushlike body,
sparse branching, dichotomous and nonastomosing at base. All tubules, as suggested by the group name,
are divided internally by septa with foramina (Tendal & Hessler, 1977) (Plate 4).
Edgertonia spp. and Komoki mudball spp. these are the dominant benthic Foraminifera in the Porcupine
Abyssal Plain samples. In addition to the above mentioned Komokiacea, a number of other
monothalamous Foraminifera (Muddy Tube spp., Lagenammina spp., Rhizammina spp.) and
polythalamous (Reophax spp.) Foraminifera were studied in the Antarctic samples.
Result Molecular work
Sixty-seven samples from Porcupine Abyssal Plain (NE Atlantic Ocean) and 24 samples from Weddel
Sea (Antarctica) were analysed using molecular approach. It was possible obtain only DNA sequences
from fragment of the SSU rRNA gene of monothalamous agglutinated Foraminifera (Astrorhiizina) and
polithalamous agglutinated and calcareous Foraminifera (Textulariina and Rotaliina). Only 7 DNA
sequences from komokiacean morphotypes were obtained and results are represented in figure 3 (a-b).
We obtained also DNA sequences of monothalamous and polithalamous agglutinated Foraminfera
(Lagenammina spp., Reophax spp. and Rhizammina spp.) from the PAP; this is an important goal
because all data of this genera come from shallow water or polar settings. In this case we have the first
DNA sequences from deep Atlantic Ocean.
Discussion and Conclusion
Komokiacea are an important group of macrofaunal sized agglutinated Foraminifera. This group
of protists eluded, until recently, description because fragility of many species means that they were
often lost during sample processing or their identification and classification were difficult. Even when
present in sample residues, they are difficult to recognize as organisms (Tendal, 1979), looking instead
like non descript organic detritus, poorly washed balls of sediment, or minor fragments from the surface
of some unidentified larger organisms (Tendal & Hessler, 1977) (Gooday & Cook, 1984).
Very few observations have been made on live cells of Komokiacea although they are classified
within the Foraminifera, granulo-reticulate pseudopodia have not been observed in any true
komokiacean organisms, causing some workers to doubt their assignment to this group. The group has
non clear ultrastructural identity and is distinguished by the unusual gross morphology consisting of a
system of branching of tubules.
Usually foraminiferal species are identified exclusively on the basis of morphological characters,
such as the number and form of the chambers, the form of test periphery and the type of ornament.
However, the paucity of morphological characters and their relatively large variability renders species
identification in Foraminifera and in Komokiacea particularly difficult. In this project we therefore
made the first attempts to investigate what Komokiacea are and their phylogenetic position between
Foraminifera group based on molecular data. Our results are based principally on analysis of ribosomal
DNA sequences.
During the course of this project we met problems with PCR analyses and on primers use. PCR
is a tool of unrivalled power, but as is so often the case, this power is linked to unrivalled complexity.
The source of this complexity is the PCR reaction itself-a myriad of ionic interactions, kinetic constants,
and enzymatic activities, all taking place repeatedly and, hopefully, perfectly, within the space of a few
hours. Because each DNA (template) and each primer pair is different, and because molecular
systematics tend to use a wide variety of taxa or primers in a single research program, PCR reactions
need to be carefully optimised. This means that a certain amount of trial and error is an integral part of
the PCR experience. Apart from PCR conditions attention has been focused on the primers and how
strongly they anneal to the template DNA. A first series of analyses have been made using foraminiferal
primers and in particular primers with monothalamous foraminiferal target. We obtained few results
associated to specimens belonging to polythalamous agglutinated (Reophax) and calcareous
(Epistominella) Foraminifera. A second series of analyses were made using universal eukaryotic
primers obtaining only contaminant sequences of animal and fungi. Other studies will be made using
other foraminiferal primers. There is no evidence yet, but the possibility exists that these results could
lead us to not consider Komokiacea as Foraminifera.
On the other hand morphological features, wall composition and test structure all suggest that
Komokiacea are Foraminifera.
The test of Komokiacea are partially formed organically but it also include a varying proportion
of extraneous matter as the typical foraminiferal agglutinated tests. The foreign particles utilized in test
construction could reflect to some extent the local environment; material commonly utilized includes
quarts grains, various heavy minerals, clay or carbonate fragments or grains, and organic debris,
including tests of smaller Foraminifera, radiolarians, coccoliths, fragments of molluscan shells, and
sponge spicules (Loeblich & Tappan, 1964). However Komokiacea are known to accumulate very fine
particles within tubule lumen where they are presumably digested (Gooday & Cook,1984). Many
aspects of the ecology and biology of the Komokiacea are poorly understood: protoplasmic body,
nucleus, pseudopodia, gamets, cysts and in particular symbionts and parasites life history and feeding
habits are still unknown.
Little is known also about the distribution of komokiacean species (Schroder et al., 1989; Gooday
& Cook, 1984; Kamenskaya, 1993).
Systematic tree obtained from molecular data (figure 3) revealed the distribution of several
komokiacean DNA sequences with environmental DNA sequences or not common foraminiferal
DNA sequences in a clade of monothalamous agglutinated Foraminifera from several habitats. These
DNA sequences could belong to parasite organisms living in the sediment particles agglutinated by
Komokiacea. This strategy is common to several protozoans and from the literature we know that the
coccidian Trophosphaera planorbulinae (LE CALVEZ) (Loeblich and Tappan, 1964) lives in the larger
chambers of Elphidium crispum extending throughout the test and destroying it eventually. Nematode
worms may also be parasitic on them. An other condition is represented by bryozoan colonies that inter
grow as networks with the komokiacean tubules (Lana and Edgertonia genera) (Gooday & Cook, 1984)
forming a symbiotic relation between ctenostome peristomes of Bryozoa and the tubule mass of
Komokiacea. These kinds of relationships complicate the study of komokiacean molecular
identification.
Our findings suggest the following conclusions 1) to find DNA sequences of Komokiacea is
necessary perform the PCR analyses with new foraminiferal primers or other universal primers; 2) we
need to improve our knowledge of ultrastructural morphology in order to demonstrate that Komokiacea
are really Foraminifera; 3) we need more information about the natural history of Komokiacea in
general.
Figure Captions
Fig. 1 Location Map Antarctic Samples
Fig. 2 Location Map NE Atlantic Samples
Fig. 3 Molecular data a) 462 sites were used for the analysis with sB primer; phylogenetic
analyses were performed with the neighbor joining method (NJ) using Kimura 2 distance algorithm;
b) 100 sites were used for analysis with s17 primer; phylogenetic analyses were performed with the
neighbor joining method (NJ) using Kimura 2 distance algorithm.
Plates of Komokiacean Foraminifera from NE-Atlantic
Plate 1 Chain-like komokiaceans: 1) Chain sp.1 - 2) Chain sp.2 - 3) Chain sp.3 4) Chain
sp.4 5) Chain sp.5 6) Chain sp.6 7) Chain sp.7
Plate 2 - Komokia-like group: 1) Komoki sp.1 2) Komoki sp.2 3) Komoki sp.3 4) Komoki
sp.4 5) Komoki sp.5 6) Komoki sp.5-bis 7) Komoki sp.6 8) Komoki sp.7
Plate 3 - Lana group: 1) Lana mudball 2) Lana sp.1 or Cerebrum (Schroder) 3) Lana sp.2
4) Lana sp.3 5) Lana sp.4 6) Lana sp.5 7) Lana sp.5a 8) Lana sp.6
Plate 4 Other Komokiacean Foraminifera: 1) Ipoa spp. 2) Septuma spp. 3) Reticulum sp.1
4) Edgertonia argillispherula 5) Edgertonia floccula 6) Mudball Komoki sp.2 7) Mudball
Komoki sp.3 8) Mudwall Crithionina spp
Plate of Komokiacean Foraminifera from Weddel Sea
Plate 5 Other Komokiacea Foraminifera: 1) Ipoa spp. 2) Septuma spp. 3) Normanina spp.
4) Edgertonia spp. 5) Saccorhiza spp.
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A
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Figure 1. - Location Map
Phylogenetic Tree - sF1-sB primers
0.034
Epistominella
Anna4941
SV2647 Reophax
RIS3889
RIS3883 3
SV E9 R.scorpiurus
1398 Reophax
Anna 4945
A68 231 Reophax
A324 241 Reophax
RIS3884
1212
1018 Psammosphaera
TB3929 Psammosphaera
A26 Silver
W274 Saccammina
RIS3907 silver
TJ528 H.alba
GU1764 H.alba
K33 Cylind
3999 213 Dunstaff
GU1780 argenteus
JP M5
GU1784 B.flexilis
4004 226 Dunstaff
Edaphoallogromia
Anna 4951
AllogromA
A.latticolaris
GU1760a
4006 228 Dunstaff
GU1760 B.minuta
A2919 Hyperammina
A1037_Crithionina
W37a Vanhoeffenella
Anna4947
W178 Vanhoeff
SV2857 H.hirudinea
A1157 H.hirudinea
Boderia
Anna4943
RIS3923 Q Komoki
GU1776 P.aurata
JP M31
HAB CR2 3cl
A1124 Hippocrepina
JP P4
Anna3502 B
Quartzball
Notodendro
1045 Rhabdammina
1439 H.bradyi
SV2844 mudball
2359 Psammophaga
K51 P.simplora
TB3790 Psammophaga
4023 262 Dunstaff
TJ563 Saccodendron
TJ547 Armorella
A.rara
A.triangularis
613 Reticulomyxa
Lana sp.1
Komoki sp.3
Normanina
Chain sp.1
Reophax spp.
Epistominella exigua
0.026
Epistomine
Anna4941
Sv2647 Reophax
A68 231 Reophax
A324 241 Reophax
RIS3884
1212
1018 Psammosphaera
RIS3889
1398 Reophax
Anna4945
SV E9 R.scorpiurus
Tb3929 Psammosphaera
Gu1780 argenteus
JP M5
Gu1784 B.flexilis
4004 226 Dunstaff
Anna3498 17
Anna3496.1 17
Anna3496.2 17
Anna3455.4 17
Anna3397.4 17
Anna3455.3 17
Anna3455.6 17
SV2841
Sv2840 10
W274-Saccammina
A26 Silver
Tj528 H.alba
Gu1764 H.alba
Anna3502 B
Anna3502 17
k33-Cylind
3999/213/Dunstaff
RIS3923 U2
Anna3315 17
A2922 Tinogullmia
Anna4943
Boderia
RIS3923/Q/Komoki
GU1776_P.aurata
JP M31
Quartzball
Notodendro
1439 H.bradyi
1045 Rhabdammina
Sv2844 mudball
Edaphoallogromia
Anna4951
AllogromA
A.latticolaris
4006/228/Dunstaff
GU1760a
GU1760-B.minuta
A2919 Hyperammina
A1037 Crithionina
W37a Vanhoeffenella
Anna4947
W178 Vanhoeff
2359 Psammophaga
k51-P.simplora
Tb3790 Psammophaga
4023/262/Dunstaff
Anna3474 17
A.rara
A.triangularis
Tj563 Saccodendron
Tj547 Armorella
Sv2857 H.hirudinea
A1157 H.hirudinea
Anna3310
A1124 Hippocrepina
JP P4
613 Reticulomyxa
Lana-like, Antarctica
Contaminants, Antarctica
Lana sp.1, NE Atlantic
Normanina, Antarctica
Chain sp.1, NE Atlantic
Komoki sp.3, NE Atlantic
Normanina, Antarctica
Lana-like, Antarctica
Phylogenetic Tree - sF1-s17+sB primers
Epistominella exigua, NE Atlantic
Reophax spp., NE Atlantic
1 2
3 4
5 6
7
Plate 1 - Chain-like komokiaceans
1
2
4
3
5
6
7
8
Plate 2 - Komokia-like group
1 2
3
4
5 6
7
8
Plate 3 - Lana group
1
2
3
4
5
6
7
8
Plate 4 - Other Komokiacea Foraminifera
Plate 5 - Komokiacean Foraminifera from Weddel Sea
1 2
3
4 5