J. Euk. Microbiol., 40(3), 1993, pp. 272-287
0 I993 by the Society of Protozoologists
Marine Heterotrophic Amoebae, Flagellates and Heliozoa from Belize
(Central America) and Tenerife (Canary Islands), with Descriptions of
New Species, Lufisphaera bulboehaete N. Sp., L. longihastis N. Sp.,
L. turriformis N. Sp. and Paulinella intermedia N. Sp.
N A J A VBRS
Institut for Sporeplanter, University of Copenhagen, DK-1353 Copenhagen K., Denmark’
ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established
from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the
choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids,the bicosoecids, the chromulinids, the pedinellids and a variety
of taxa of uncertain affinities (Protista incertae sedis). The identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and
S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described one new species of the amoeba genus
Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Lumphaera Belcher & Swale, 1915 (Lumphaera
bulbochaete n. sp.; L. longihastis n. sp.; L. turriyormis n. sp.).
Supplementary key words. Biogeography, Choanoflagellida, Cryptophyceae, diversity, Euglenozoa, incertae sedis taxa, protists,
protozoa, Straminopiles, taxonomy.
D
URING the last decade much effort has been put into
describing the diversity of marine heterotrophic flagellates
and the biology of individual species [ 18, 27,45, 65, 741. These
investigations may lead not only to a better understanding of
the ecological role of the heterotrophic protists [70], but also to
resolution of many problems pertaining to protist relationships
[421.
A large number of the heterotrophic protists encountered in
marine habitats are still undescribed, or not yet examined using
modem techniques [48]. Additionally, knowledge of the distribution of named species is still limited. This paper presents the
results of studies on heterotrophic protists, particularly flagellates, in crude cultures and bacteria-enriched cultures established from water samples from Belize (Central America) and
from Tenerife (Canary Islands).
Due to the rapidly increasing molecular and ultrastructural
database [e.g. 1, 16, 441 our view of protist evolution and phylogeny is changing dramatically. The boundary of Protista is in
dispute and taxonomic schemes should be applied with caution
until a more consistent view is obtained [e.g. 531. The protists
encountered in the present study are therefore dealt with under
colloquial headings: amoebae, heliozoa, flagellates and the incertae sedis group. Each of these groups are subsequently divided into a number of taxa over which there is little debate.
For the flagellates the taxa would be Choanoflagellida (Acanthoecidae, Salpingoecidae), Cryptophyceae, Euglenozoa (Euglenophyceae, Kinetoplastida) and “Straminopiles” (BicosoeI Current address: School of Biological Sciences, University of Sydney
A08, NSW 2006 Australia.
cales, Chromulinales, Pedinellales). The incertae-sedis group
accommodates taxa of uncertain taxonomic position, presently
a vast number of genera [e.g. 481. The taxa are treated under
the International Code of Botanical Nomenclature (ICBN) [ 171
or the International Code of Zoological Nomenclature (ICZN)
[ 5 11 according to previous practice/tradition. Thus the ICZN is
applied to the majority of the taxa, except Cryptophyceae, Euglenophyceae, Bicosoecales, Chromulinales and Pedinellales,
which traditionally are subject to the provisions of the ICBN.
This approach to the nomenclature of the protists reflects their
phylogeny as presently conceived, whereas the application of
either code to all protists would be artificial and confusing (see
Patterson & Larsen [46,47] for an account of the nomenclatural
problems associated with the “ambiregnal” protists).
MATERIAL AND METHODS
Seawater samples were collected at the following sites around
Carrie Bow Cay, Belize, Central America (1 8” 48‘ N; 88” 05’
W): The back reef and the pier at C a m e Bow Cay, Twin Cays
Channel, Hidden Creek (Twin Cays), Hidden Lake (Twin Cays)
and Man of War Island. C a m e Bow Cay is a coral reef island,
whereas Twin Cays and Man of War Island are intertidal mangrove islands. The samples were collected in acid-washed 10liter plastic buckets over the side of a small boat by Jacob Larsen
and 0jvind Moestrup, February 2-March 2, 1990. Temperature
and salinity were within the range of 28” to 30.5” C, and 32 to
34% S respectively. Two to five liters of each sample were
concentrated over a 2-pm Millipore filter by gravity filtration,
transferred to 20-ml glass test tubes, and enriched with 34% S
modified Erdschreiber medium. At Carrie Bow Cay the crude
Fig. 1-13. Heterotrophic protists from Belize and Tenerife. 1. Transmission electron micrographs of shadowcast whole mount of the test of
Paulinella intermedia n. sp. from Tenerife. Bar = 1 pm. 2-9. Light microscopy, phase contrast optics, of living cells. 2-5. Salpingoeca camelopardula
from Belize, growing in the empty thecas of Tetraselmis. Arrows point to the contours of the Salpingoeca thecas. Notice that the protoplasts do
not fill out the thecas completely, and that the cells are bilaterally, not radially symmetrical (compare 2 with 3 and 4 with 5). 2. Cell with long,
thin neck showing beating flagellum. Bar = 5 pm. 3. Cell with long, thick neck. Bar = 5 pm. 4. Cell with short, conical neck. Bar = 5 pm. 5.
Small cell with short, thin neck. Bar = 5 pm. 6-9. Salpingoeca ringens from Belize. 6. Sessile cell showing anterior margin of theca (arrow). Bar
= 5 pm. 7. Sessile cell. Arrows point to the contours of the theca. Bar = 5 pm. 8. Sessile cell showing the posterior thecal bulb (arrows). 9. Freeswimming cell showing the posterior pseudopodia (long arrows). Pedicels and bulbs on vacated thecas are also visible (small arrows). Bar = 5
pm. 10-13. Transmission electron-micrographsof shadowcast whole mounts. 10. Salpingoeca ringens, complete cell showing the bulb (small
arrow) and the fibrous pedicel (long arrow). Bar = 5 pm. 11, 12. Salpingoeca camelopardula. 11. Detail of fig. 12 showing the amorphous material
of the theca. Bar = 0.5 pm. 12. Complete cell showing the asymmetrical cell shape. Bar = 5 pm. 13. Salpingoeca ringens. Detail of empty theca
showing the thecal chamber, the neck and the “bulb” (small arrow), which is composed of amorphous material. Notice that the thecal neck of
empty thecas may not be flared (large arrow). Bar = I pm.
272
V0RS-(SUB)TROPICAL HETEROTROPHIC PROTISTS
273
274
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
VQRS-(SUB)TROPICAL HETEROTROPHIC PROTISTS
cultures (23 in all) were kept in the light, but at a temperature
below 25" C. The cultures were transported to the Institut for
Sporeplanter (University of Copenhagen) after a maximum period of four weeks.
A single seawater sample was collected in a 1-liter mineral
water bottle at the beach near Callao Salvaje, Adeje, SW Tenerife, Canary Islands (28" 10' N; 17" 50' W), by Kristian M. Christensen and Susan Rasmussen November 22, 1988. Temperature
and salinity were ca. 20°-22" C, and ca. 349x1S respectively. The
sample was immediately transported to the Institut for Sporeplanter.
In order to nourish bacterivorous heterotrophic flagellates,
bacteria-enriched subcultures were established from the crude
cultures from Belize and from the water sample from Tenerife.
A bacterial enrichment culture was prepared from 3 4 9 ~
S ~medium, two wheat grains, one ml of bacterial suspension (Vibrio
natriegens, kindly provided by A. M. Onarheim, University of
Bergen) plus a few milliliters of inoculum. These cultures were
maintained in Nunclon-Delta 50-ml tissue culture flasks (Life
Technologies, European Division) at 20" C, and under a light :
dark regime of 16:8 hours (light quantity: 42 pmol/m2/s). All
of the cultures studied were mixed cultures (uncloned populations).
Light microscopical observations were made on an Olympus
BH2 microscope. Electron microscopical whole mounts were
made as described by Moestrup and Thomsen [35] and Thomsen [63]. Some grids were stained with uranyl acetate as described by Moestrup [34] and some were shadowcast with gold/
palladium. The grids were viewed on a JEOL JEM-100 SX
electron microscope at the Institut for Sporeplanter.
RESULTS
The species of heterotrophic protists recorded and the sites
at which they were found are listed in Table 1. Some of the
species are illustrated in Fig. 1-35. Detailed accounts of the
morphology, ultrastructure and taxonomy of many of the taxa
listed here have already been given by Larsen & Patterson [27],
Page [40], Patterson & Larsen [45], Siemensma [54] and Vsrs
[72]. Only taxa that have not previously been dealt with by these
authors, that deviate from the existing descriptions, or that are
novel will be dealt with in the following. Tables 2-5 summarize
sizes of selected species. Data on size listed in the text below is
followed by a parenthesis indicating size figures extracted from
the type descriptions or the cited literature.
A parenthesis following each higher order taxon indicates
whether the nomenclature follows the botanical (ICBN) or zoological codes (ICZN). When a botanical name is used the equivalent zoological name is also indicated.
AMOEBAE
Many different amoebae inhabit marine waters. They may be
as common as heterotrophic flagellates or under certain circumstances, even more abundant [7, 8, 191. A few species of naked
275
and testate amoebae were encountered during this study. One
of these could not be identified, but its description has been
included to demonstrate the diversity ofthese heterotrophs. Due
to the uncertainty of the classification of amoebae [39, 411, a
complete listing of the higher order taxa is omitted.
FILOSEA Leidy, 1879 [ICZN]
Paulinella Lauterborn, 1895
Testate amoebae with internally formed siliceous scales. The
scales are rectangular or elongate hexagonal plates, which are
arranged in longitudinal columns (usually five) [22, 281.
Paulinella intermedia Vsrs, n. sp.
(Fig. 1)
Size of test. Ca. 2.9 x 6.6 pm; neck diam.: ca. 1.9 pm.
Diagnosis. Entire test composed of flat, rectangular plate
scales with rounded comers.
Type specimen. Collected at Callao Salvaje, Adeje, Tenerife
(= specimen in Fig. 1).
Description. Only the test has been observed. The test consists of an ovate chamber with an apical short neck, and is
composed of scales. The scales are flat, slightly bent, rectangular
plates with rounded comers. The scales are arranged in columns
parallel to the long axis of the test. There are 3-4 columns and
6-7 scales in each column. A few smaller scales are found at
the posterior end of the test. The neck of the test is composed
of three scales, which are narrower than those of the test chamber.
Remarks. The test of Paulinella ovalis (Wulff) Johnson et al.
[22] is composed of five columns of 5-6 thick, curved and ridged
scales. There are a few thick scales at the posterior end, and
three flat neck-scales. The average size of the test is 3 x 4.5
pm, with a neck diameter of ca. 1 pm. Newly formed scales are
also thick and curved [22]. Paulinella intermedia is similar to
P. ovalis in size, and in the number of scales composing the test,
but it is distinguished by possessing flat scales only.
Paulinella intermedia is too small to be referred to Paulinella
chromatophora Lauterborn, 1895 which measures 17-22(-35)
pm in length and 15-20 pm in width, with a neck diameter of
ca. 5 pm. The test comprises 5-6 columns of 9-12 scales [22,
24, 281.
Pseudodiflugia Schlumberger, 1845
Testate amoebae with rigid test composed of proteinaceous
material with agglutinated particles. The test has a single, terminal aperture [ 5 , 381.
Pseudodiflugia sp.
(Fig. 34A)
Size. Test diam.: ca. 10 pm; filopodial length: 10-1 5 pm.
Description. The test is spherical, yellow or orange, and has
an irregularly ornamented or creased surface, possibly from attached pieces of detritus. The test has a narrow, circular neck
t
Fig. 14-24. Heterotrophic protists from Belize; transmission electron micrographs are of stained whole mounts (16, 18, 24), all other figures
are micrographs of shadowcast whole mounts. 14. SavilIea micropora.A completecell and an empty lorica. Bar = 2 pm. 15. Heierophrys myriapoda.
Detail showing a bundle of the organic spicules that surround the cell. Bar = 1 pm. 16. Cafeteria minuta, with a long hairy flagellum. Bar = 2
pm. 17. Cafeteria roenbergensis, with a relatively short hairy flagellum. Bar = 1 pm. 18-24. Paraphysomonas spp. 18-21. Paraphysomonas
aniarctica.18. A typical, long-spined scale. Bar = 0.5 pm. 19. A short-spined scale. Bar = 0.5 pm. Arrows point to the abruptly changing thickness
of the spines (the transition zone between the main part of the spine and the spine tip). 20. Complete cell showing variation in the morphology
of the scales. Small arrow points to a small scale and the large arrow to a spineless scale. Bar = 1 pm. 21. Cell with very a long, hairy flagellum
( 1 3 pm) and numerous long-spined scales. Small arrow points to a short-spined scale. Bar = 1 pm. 22-24. Paraphysomonus imperforata.22. An
aberrant scale with abruptly changing spine thickness (arrow). Bar = 0.5 pm. 23. A typical scale with a gradual change in the thickness of the
spine (arrow). Bar = 0.5 pm. 24. Complete cell showing typical scale morphology. Bar = I pm.
276
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
Table 1. Heterotrophic protists encountered in marine cultures from the Came Bow Cay area (Belize, Central America) and Callao Salvaje,
Adeje (Tenerife, Canary Islands). Species records from arctic waters [711, temperate waters [72, 731 and tropical sediments [27] are added for
comparison. The data are listed as observations in freshly collected water samples, or EM whole mounts made from these (o),or in enrichment
cultures (x).
Tenefife
Belize localities
Taxa of heterotrophic protists
AMOEBAE
FILOSEA Leidy, 1879
Paulinella intermedia Vers n. sp.
Pseudodiflugia Schlumberger, I845 sp.
LOBOSEA Carpenter, 1861
Yanella aberdonica Page, 1980
1
2
“STRAMINOPILES”
BICOSOECALES Grass6 1926
(= BICOSOECIDA Grass6 & Deflandre, 1952)
Bicosoeca kepneri Reynolds 1926 or
Bicosoeca lacustris James-Clark 1867
Cafeteria minuta (Ruinen) Larsen & Patterson 1990
Cafeteria roenbergensis Fenchel & Patterson 1988
Pseudobodo trernulans Griessmann 1913
CHROMULINALES Pascher 19 12
(= CHROMOMONADINA Webs, 1893)
Ochromonas Vysotskii 1887 or
Spurnella Cienkowski 1870 sp.=
Paraphysomonas antarctica Takahashi 1987
Paraphysomonas imperforata Lucas I967
PEDINELLALES Zimmermann et al. 1984
(= CILIOPHRYIDA Febvre-Chevalier, 1985)
Actinomonas rnirabilis Kent 1880 or
Pteridomonas danica Patterson & Fenchel 1985d
INCERTAE SEDIS TAXA
Ancyromonas sigmoides Kent, 1880
Bordnamonas tropicana Larsen & Patterson, 1990
Lufiphaera bulbochaete Vers n. sp.
Lufiphaera longihastis Verrs n. sp.
Lufiphaera turriformis Vers n. sp.
Metopion fluens Larsen & Patterson, 1990
Telonema subtile Griessmann, 1913
4
5
6
0
X
X
HELIOZOA
CENTROHELIDA Kiihn, 1926
Heterophrys myriapoda Archer, 1869b
FLAGELLATES
CHOANOFLAGELLIDA Kent, 1880
SALPINGOECIDAE Kent, 1880
Salpingoeca camelopardula Noms, 1965
Salpingoeca ringens Kent, 1880
Salpingoeca tuba Kent, 1880
ACANTHOECIDAE Noms, 1965
Diaphanoeca grandis Ellis, 1930
Savillea micropora (Noms) Leadbeater, 1975
Stephanoeca dipl. paucicostata Throndsen, 1969
CRYPTOPHYCEAE Fritsch 1927
(= CRYPTOMONADIDA Senn, 1900)
Goniomonas amphinema Larsen & Patterson 1990
Goniomonas paci9ca Larsen & Patterson 1990
Goniomonas truncata (Fresenius) Stein 1878
EUGLENOZOA Cavalier-Smith, 1981
EUGLENOPHYCEAE Schoenichen 1925
(= EUGLENIDA Biitschli, 1884)
Petalomonas pusilla Skuja 1948
KINETOPLASTIDA Honigberg, 1963
Bod0 designis Skuja, 1948
Bodo parvulus Griessmann, 19 13
Bod0 saliens Larsen & Patterson, 1990
Bod0 sultans Ehrenberg, 1832
3
Arctic
___ locali7’
ties
X
X
X
x
x
X
0
X
X
X
X
x
X
ox
x
X
X
X
X
X
X
X
X
X
x
x
x
x
X
x
x
x
x
x
x
X
x
X
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
X
X
x
X
X
X
X
X
X
X
X
ox
X
X
X
X
X
X
ox
X
X
ox
ox
X
X
X
ox
X
X
X
X
ox
X
ox
X
X
ox
X
X
X
x
x
x
Tropical
localities
X
x
X
x
x
Ternperate
localities
x
X
X
X
X
X
ox
X
X
X
X
X
X
X
X
ox
ox
X
277
V0RS -(SUB)TROPICAL H ETEROTROPHI C PROTlSTS
Table 1. Cont.
7”
Arctic
localities
Ternperate
localities
Tropical
localities
X
X
ox
X
Tene.-...
rife
Belize localities
e
Taxa of heterotrophic protists
1
2
3
APUSOMONADIDAE Karpov & Mylnikov, 1989
Amastigomonas debruynei De Saedeleer, 1931
4
5
6
X
_
_
a Localities: 1, Came Bow Cay back reef; 2, Came Bow Cay pier; 3, Twin Cays Channel; 4, Hidden Creek 5, Hidden Lake; 6, Man of War
Island; 7, Callao Salvaje, Adeje.
(Fig. 15, 33M).
c The genera Ochromonas and Spumella cannot be distinguished if the chloroplast in Ochromonas is not conspicuously present [50, 721.
* Actinomonas mirabilis and Pteridomonas danica can only be reliably identified by ultrastructural studies of the flagellar apparatus [72].
surrounding an opening from which a few smooth, thin and
often branched pseudopodia (filopodia/reticulopodia)may protrude. The test is opaque and obscures the view of the cells
inside it. The substructure of the test is not known.
Remarks. A large number of genera of testate amoebae have
been described, particularly from soil and fresh water [e.g. 5 ,
3 1, 381. The present marine specimen superficially resembles
Pseudodlfflugia gracilis Schlumberger, 1845 (diameter 2 0 4 5
pm [5, 38]), described from fresh water, but it is too small to
be referred to this species. It may represent a new species, but
a formal description is omitted pending information on the
substructure of the test. A similar organism has been observed
in southern Kattegat, Denmark (Vors, unpubl. observ.).
FLAGELLATES
CHOANOFLAGELLIDA Kent, 1880 (ICZN)
SALPINGOECIDAE Kent, 1880
Sdpingoecu James-Clark, 1867
Choanoflagellates surrounded by a single firm organic theca,
which is closed at the posterior end and which is visible in the
light microscope [30, 641. The genus accommodates more than
70 species distinguished by thecal morphology mainly at the
light microscopical level [e.g. 21, 23, 37, 57, 581. Many species
are probably synonymous and the identification of species of
Salpingoeca is therefore problematic. Furthermore, some “floras” or “faunas” have been found to contain erroneous illustrations (e.g. Lemmermann [32], see Vors [72]), and it is recommended that the type descriptions are consulted for
identifications.
Table 2 summarizes sizes of the species below measured from
10 different living cells of each species, except S. tuba (two cells).
Salpingoeca camelopardula Noms, 1965
(Fig. 2-5, 11-12, 34B, Table 2)
See description by N o m s [37].
Remarks. This species is very distinct, being the only species
with a regular, bilaterally symmetrical (not radiate) theca. The
length of the thecal neck may vary (Table 2). It always protrudes
from one end of the thecal chamber. The protoplast does not
fill out the theca completely, as noted also by Thomsen [61].
Salpingoeca camelopardula grew well in the crude cultures, where
it was found attached to empty thecas of Tetraselmis (Prasinophyceae). The neck of some cells was coiled because straight
growth was obstructed by algae that settled on top of the cells
(Fig. 34B). All parts of the S. camelopardula theca appear to be
amorphous (Fig. 1 1, 12).
Previous records (marine). California, USA [37], and Gulf
of Elat, Israel [6 11.
Salpingoeca ringens Kent, 1880
(synonym: Salpingoeca eurysioma Stokes, 1886)
(Fig. 6-10, 13, 34C, 35A, B)
Description. Theca with an ovate chamber and a broad, flaring neck. The widest part of the theca is the neck and the theca
is typically 1.5 times as long as broad. The chamber gradually
tapers towards the posterior, pedicellated end. In the transition
zone between the chamber and the pedicel, the chamber may
carry a small bulb-like swelling (visible in LM) (Fig. 8-10, 13,
Table 2. Size values (measured from living and dried cells) for the specimens ofSalpingoeca camelopardula. Salpingoeca ringensand Salpingoeca
tuba from Belize and Tenerife. Reference values from the type descriptions and previous records are listed for comparison. All numbers are
in pm.
Size of theca
~
Total length
Thecal chamber
Length
of neck
Sulpingoeca camelopardula
Almost as theca
7-20
Reference values [37]
As theca
14.5-15.5
Reference values [61]
Almost as theca
6.5-13.5
Sulpingoeca ringens
2-4 x 4-7.2
6-1 Ib
_c
ca. 12.7
Reference values [23]
Sulpingoeca tuba
ca. 3 x 10
15-20
Reference values [23]
6.4-12.7
Reference values from previous records
10-60
a Width listed first.
Exclusive of pedicel (length: 4-16 am). Pedicel length is not listed by Kent 1231.
Data not available.
2.26 x 3.24
4.7-5 x ca. 3.5
4-5.3 X 3-6
3-5 x 5-8
4-1 5
11-12
3.5-7.5
1.6-3.0
Cell size
-
4-5 x 15-20
-
5-7 x 10-20
Neck width
Smallest
Largest
0.4-2.0
0.4-0.5
1.3-2.0
3.2-4.0
3-7
ca. 2
3-5
3.9-7.2
ca. 8.5
-
~~
278
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
Fig. 25, 26. Transmission electron micrographs of shadowcast whole mounts of Luffisphaera bulbochaefe n. sp. from Belize. 25. Complete
cell showing the dense layer of base scales and the scattered spine scales, surrounding the cell body. Bar = 1 pm. 26. Detail of 25 showing the
spine and base scales. Bar = 0.5 pm.
pingoeca eurystoma Stokes, 1886 (Fig. 35A) is a junior synonym
of S. ringens, as suggested by Zhukov & Karpov [76], since the
characteristics of this species are identical with those of S. ringens [ 5 8 ] .
In the closely related species Salpingoeca inquillata Kent,
1880 (Fig. 35C), the widest part of the theca is the thecal chamber and the length of the theca is two times the width. However,
this is not well illustrated by Kent, and his drawings of S. inquillata and s. ringens are much alike (Fig. 35B,C). A similar
species, Salpingoeca curvipes Kent, 1880 (Fig. 35D), is distinguished by the curvature of the pedicel. This character is not
stable. The theca or stalk of sessile choanoflagellates may be
straight or bent, depending on the nature of the habitat. A coiled
theca and bent stalks have been observed in specimens of S.
camelopardula (see above) and S. ringens, respectively. Kent
[23] also uses the shape of the protoplast (ovate as opposed to
flask-shaped) as a specific character. However, the shape of the
protoplast, the degree to which it fills out the theca, and its
small arrows). All parts of the theca appear to be amorphous
(Fig. 13), except the distal part of the pedicel which is fibrous
(Fig. 10, long arrow). At the time when this population was
observed, most of the protoplasts were situated in the anterior
part of the thecas. The protoplasts may leave the theca and
swim freely. Vacated thecas may be seen in detritus (Fig. 9,
small arrows). Free-swimming cells may have thin pseudopodia
protruding from the cell posterior, giving them a spiny appearance (Fig. 9, long arrows). This is also typical of free-swimming
cells of Monosiga and Codosiga [72].
Remarks. The observations on the present specimens are
generally in accord with Kent’s description of Salpingoeca ringens (Fig. 35B), although the theca of S. ringens sensu Kent [23]
lacks the bulb. However, because the bulb may be absent or
weakly developed in the present species, they are regarded as
identical. According to Kent, the distinguishing characters of
this species are that the widest part of the theca is the anterior
neck, and the length of the theca is 1.5 times the width. Sal-
Table 3. Size values (measured from dried cells) for the specimens of Paraphysomonas antarcfica and Paraphysomonas imperforafa from
Belize. Reference values [59] are listed for comparison. All numbers are in pm.
Spine scales
Cell
diameter
Paraphysomonas antarctica
Reference values [59]
Paraphysomonas imperforata
Reference values [59]
1.0-3.1
2.0-4.3
1G 2 . 0
1.7-5.1
Flagellar lengths
Long
Short
5-13
12.5-27
5.5-9.0
1.5-3.5
1.84.5
1.0-2.5
-
-
Spine lengths
Base plate
diameter
Total
Tip
Tip/total
0.6-1.4
0.9- 1.7 5
0.5-0.9
0.4-0.97
0.42-2.25
1.0-3.25
0.7-1.4
0.7-1.13
0.15-0.32
0.3-0.7
0.5-0.8
0.4-1 .o
1/14-1/2.2
1/4.6
1/2.1-111.7
1/2-1/ 1.7
Ve)RS-(SUB)TROPICAL HETEROTROPHIC PROTISTS
279
Fig. 27-32. Transmission electron micrographs of shadow cast whole mounts of Lufiphaera Iongihastis n. sp. and Lufiphaera turriformis
n. sp. from Belize. 27-29: Lufiphaera longihastis n. sp. 27. Complete cell showing the dense layer of base scales, and the scattered spine scales,
surrounding the cell body. Bar = 1 pm. 28. Detail of 27 showing individual spine and base scales. Bar = 0.5 pm. 29. Individual spine scale. Bar
= 0.5 pm. 30-32. Lufiphaera turriformis n. sp. 30. Complete cell showing the dense layer of base and spine scales, surrounding the cell body.
Bar = 1 pm. 31. Detail of 30 showing individual spine scales and base scales (long arrows). Notice also the finely striated rods of the spine scales
(small arrow). Bar = 0.5 pm. 32. Individual spine scale showing the striated distal thin rod (arrow). Bar = 0.5 p m .
280
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
A
b
d
B
a b
d
C
a b
c d
e
f
e
e
f
f
Fig. 33. Heterotrophic protists from Belize. Drawings of spine scales of the three new species of Lumphaera. The labels (a-f) indicate the
different parts of the spines: Proximal (a) and distal (b) lattice of cylindrical base, proximal (c) and distal (d) lattice of cone or dome-shaped part,
narrow rod or tip of cone/dome (e), distal rod or spine (0. A. Lumphaera bulbochaete n. sp. B. Lumphaera turriforrnis n. sp. C . Lujjisphaera
longihastis n. sp.
position in the theca, probably varies according to the age or
physiological state of the cell. This is evident from the work of
Leadbeater [29] on Proterospongia choanojuncta (as the Choanoeca perplexa stage), and from the published drawings of Salpingoeca gracilis [9, 12, 601.
The three species, S. curvipes, S. inquillata and S. ringens,
thus do not seem well separated, and they were regarded as
synonymous by Boucaud-Camou [4]. However, S. ringens should
be excluded from this synonymy, since it is specifically described
as having different thecal dimensions.
Previous records (marine). England [23].
Salpingoeca tuba Kent, 1880
(synonyms: Salpingoeca cylindrica Kent, 1880;
Salpingoeca petiolata Kent, 1880)
(Fig. 34D, Table 2)
See description by Kent [23].
Remarks. Salpingoeca tuba Kent, 1880 and Salpingoeca cylindrica Kent, 1880 cannot be distinguished [23]. Salpingoeca
tuba is also similar to Salpingoecapetiolata Kent, which is characterized by the protoplast possessing a distinctive posterior
stalk. The presence or absence of a pseudopodial stalk is not a
stable character. The published drawings of S. gracilis [9, 12,
56, 601, clearly show the protoplasts of this species entirely
without or with one or more stalks (pseudopodia). Also Ellis
describing S. megacheila [ l I] and Stokes describing S. eurystoma [58], both noted that a posterior stalk (“anchoring filipode”
[ l 11) may be absent or present in the same species. The pro-
duction of posterior pseudopodia is likely to depend on the size
of the theca and of the protoplast. It thus seems justifiable to
regard S. tuba, S. cylindrica and S. petiolata as synonyms, in
concurrence with Boucaud-Camou [4]. The three species have
the same status, but Boucaud-Camou chose S. tuba as the senior
synonym.
Nonis [37] (as Salpingoeca vaginicola) reports the basal part
of the theca of S. tuba being expanded or fringed with filamentous threads. Basal filamentous threads are known from other
sessile choanoflagellates (Salpingoeca amphoridium [72] and P.
choanojuncta [29]) and their presence may be a characteristic
feature of all attached salpingoecids. Their development is probably substrate dependent [37].
Salpingoeca vaginicola Stein, 1878, is superficially similar to
S. tuba, but this species has a pointed thecal base with a pedicel.
Previous records (freshwater). England (as S. gracilis (plate
VI, Fig. 32) [23]; Germany [6]; Hungary, [15].
Previous records (marine). England [ 10,231(as S. vaginicola);
California, USA (as S. vaginicola) [37]; France [4].
CRYPTOPHYCEAE Fritsch 1927 (ICBN)
(= CRYPTOMONADIDA Senn, 1900)
Goniomonas Stein 1878
Cryptophycean flagellates possessing an anterior transverse
ring or line of ejectisomes and lacking a plastidial complex and
tubular, flagellar mastigonemes [20, 26, 271.
All named species of this genus, Goniomonas amphinema
Larsen & Patterson 1990 (Fig. 34E), Goniomonas pacifica Lar-
V0RS-(SUB)TROPICAL HETEROTROPHIC PROTISTS
28 1
Fig. 34. Drawings of some of the heterotrophic protists. A. Pseudodiflugia sp. from Tenerife, showing spherical test with extruding thin
pseudopodia. Bar = 3 pm. B. Salpingoeca carnelopardula from Belize, two cells settled in empty thecas of Tetruselmis. Bar = 5 pm. C . Salpingoeca
ringens from Belize, two cells with and without “bulbous” base of the thecal chamber. Bar = 5 pm. D. Salpingoeca tuba from Tenerife, a complete
cell and an empty theca. E G .Swimming cells of different species of Goniomonas.E. Goniomonas umphinema from Tenerife, two cells. Bar =
5 pm. F. Goniomonas paci$ca from Belize, two cells. Bar = 5 pm. G ( 1 4 ) . Goniornonas truncata from Belize, showing size range of cells. Bar =
5 pm. H-J. Cafeteria roenbergensis from Belize. H. Minute form, feeding (1) and swimming cells (2). Bar = 5 pm. I. Normal sized form, feeding
(1) and swimming cells (2). Bar = 5 pm. J. Large-celled form, feeding (1) and swimming cells (2). Bar = 5 pm. K. Cafeteria minuta from Belize,
feeding (1, 3 4 ) and swimming cells (2). The cells in 3 4 have formed pseudopodia. Bar = 5 pm. L. Puraphysomonas anturcticu from Belize,
swimming (1) and sessile, feeding cells (2). Both cells are surrounded by adhering detritus. Bar = 5 pm. M. Heterophrys myriupodu from Tenerife,
showing that the dense layer of spicules (see 15) surrounding the cell body may be visible at the light microscopical level. Bar = 10 pm. N.
Bicosoeca kepneri/lacustris from Belize, showing retracted cell in closed lorica ( l ) , and extruded, feeding cells in open loricas (2-3). Bar = 5 pm.
Beat envelope of the flagella is indicated with dotted lines.
282
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
only exception being the observations of very small cells (Fig.
34G: 1-3). These cells were otherwise typical for G. truncata.
Previous records (marine). Known from European fresh waters and from the Baltic Sea [72].
“STRAMINOPILES”
An informal group name suggested by Patterson [43] to accommodate protists with tripartite tubular hairs, and the descendants of these organisms (bicosoecids, chrysophytes, pedinellids, labyrinthulids, oomycetes, hyphochytridiomycetes,
slopalinids) (Stramenopiles as used by Patterson [43] is an incorrect spelling if the etymology of the word is respected (straminopiles is Latin for “straw-hairs”).
\
BICOSOECALES GrassC 1926 (ICBN)
(= BICOSOECIDA GrassC & Deflandre, 1952)
Biflagellate, heterokont flagellates attached in a lorica (Bicosoeca James-Clark 1867) or to a substrate (temporarily) (Caf
eteria Fenchel & Patterson 1988, Pseudobodo Griessmann 19 13)
by the tip of the posteriorly directed, smooth flagellum [50].
C
B
_b
Fig. 35. Drawings of synonymous or related species of Salpingoeca
ringens Kent, 1880. A. Salpingoeca ringens (= S. eurystoma Stokes,
1886). B. Salpingoeca ringens Kent, 1880. C . Salpingoeca inquillata
Kent, 1880. D. Salpingoeca inquillata (= S. curvipes Kent, 1880). [After
Stokes 1886 (A) and Kent 1880 (ED).] Bar = 4 pm.
sen & Patterson 1990 (Fig. 34F) and Goniomonas truncata (Fresenius) Stein 1878 (Fig. 34G), were observed in the present study
(Table 1) but only the latter species deviated from previous
descriptions.
Goniomonas truncata (Fresenius) Stein 1878
(Synonym: Monas truncata Fresenius 1858)
(Fig. 34G. 1-4)
Size. Cell length 3-10 pm (8-25 pm).
See description by Skuja [55].
Remarks. The observations on the present species (Fig. 34G:
1 4 ) are generally in accord with Skuja [ 5 5 ] and Stein [57], the
Bicosoeca kepneri Reynolds 1926/Bicosoeca lacustris
James-Clark 1867
(Fig. 34N)
Size. Lorica: 5-6 x 7-9 pm (4-10 x 10-25 pm); protoplast:
2-3 x 3-4 pm (length: 6-12 pm).
Description. Lorica ovoid or ellipsoidal and stalked. The
anterior end of the lorica is closed when the protoplast is retracted to the bottom of the lorica (Fig. 34N: 1). It is forced
open by the protoplast as it protrudes for feeding. The protoplast
is globular-ovoid, and has a conspicuous peristomal lip (Fig.
34N: 2-3).
Remarks. This species closely resembles the fresh water species Bicosoeca kepneri and Bicosoeca lacustris in overall morphology and elasticity of the lorica [2, 21, 25, 33, 571. It differs
only by being slightly smaller and because it was found in a
saline habitat. The size difference is not regarded as significant,
and other fresh water species have been recorded in salt waters
(e.g. Bicosoeca conica Lemmermann 19 14 [27]).
Bicosoeca kepneri and B. lacustris differ in the substructure
of the lorica as revealed by electron microscopical thin sections
Table 4. Size values (measured from dried cells) for selected scales, or cells, of some of the specimens of Paraphysomonas antarctica and
Paraphysornonas imperforata from Belize. All numbers are in pm.
Spine scales’
Base plate
diameter
Paraphysomonas antarctica (Fig. 20)
Small scale
Large scale
Paraphysomonas antarctica (Fig. 2 1)
Small scale
Large scale
Paraphysomonas imperforata (Fig. 22)
Aberrant scale
Paraphysomonas imperforata (Fig. 23)
Typical scale
The cells in Fig. 20, 2 1 also possess spineless scales.
Change in
Total
Tip
Tip/total
1
0.5
1.8
0.15
0.2
1/9.0
abrupt
abrupt
1
1.1
0.75
2.25
0.3 1
0.25
1/2.4
U9.1
abrupt
abrupt
0.63
1
0.5
1/2.0
abrupt
0.8
1.25
ca. 0.7
1/1.8
gradual
0.6
A single cell may possess scales in the size range given below:
Paraphysomonas antarctica (Fig. 20)
0.6-1.0
Paraphysomonas antarctica (Fig. 2 1)
1.0-1.1
Paraphysomonas imperforata (Fig. 24)
0.5-0.9
a
Spine lengths
0.5-1.8
0.75-2.25
0.7-1.3
113.3
thickness
283
V0RS- (SUB)TROPICAL HETEROTROPHIC PROTISTS
Table 5. Size values for the scales (measured from collapsed scales on dried cells) of Lumphaera bulbochaete n. sp., Lumphaera longihastis
n. sp. and Lumphaera turriformis n. sp., all from Belize. All numbers are in 0.1 pm.
Size of the different Darts of the wine scales
~~
Cylindrical base
Lumphaera
longihastis
Lumphaera
bulbochaete
Lumphaera
turriformis
a
Distal lattice
Proximal lattice
s=
h
C
Distal lattice
d
1.5-2.5 x 6.5-7.5
2-3 x 2-4
1-2 x 3-3.5‘
-d
1-2 x 3-3.5
Narrow rod
or tip of
cone/dome
e
Cone- or dome-shaped part
Proximal
lattice
2-2.5
X
2-3
1.5-2.5
X
2.5-3.5
-
6-8.5
2-3
X
2.5-3.5
-
7-8.5 x 5-7
x 4-6.5
3.5-4.5
X
ca. 1
1.5-2 x 0.5-0.8
8.5-12 x ca. 1
Distal rod
or spine
f
15-25 x 0.3-0.4
6-8.5 x 5 0.5
10-12 x 5 0.5
Width
of base
scalesb
2.5-3
3-3.5
ca. 4
See Fig. 33 for diagrammatic representation of “a” through “f’
Length not measurable.
Length listed first.
Not measurable.
[33]. The present species was not found in the EM preparations,
and thus cannot be identified with certainty.
Previous records. Both species are widespread in N. American and European fresh waters [33].
Cafeteria minuta (Ruinen) Larsen & Patterson 1990
(Synonym: Pseudobodo rninuta Ruinen 1938)
(Fig. 16, 34K)
Size. Cell diameter ca. 3 pm or measuring ca. 2 x 4 pm
(diam. 3.5-4 pm o r measuring 1-1.5 x 1.5-2.5 pm); length of
long flagellum: ca. 8.3 pm (4.5-12.5 pm); length of short flagellum: ca. 5 pm (2-5 pm).
See Larsen & Patterson [27] for a detailed description.
Remarks. This small species is distinguished from Cafeteria
roenbergensis by its longer hairy flagellum (relative to the cell
body, compare Fig. 16, 17). An extensive production of pseudopodia from all parts of the cell body was sometimes observed
(Fig. 34K: 3, 4).
Previous records (marine). Tropical Australia and Brazil [27];
W. India [52].
Cafeteria roenbergensis Fenchel & Patterson 1988
(Fig. 17, 34H-J)
Size. 2-7 x 3-15 pm (length: 3-6(-9) pm).
See Fenchel & Patterson [ 141 and Larsen & Patterson [27] for
detailed descriptions.
Remarks. Minute or unusually large specimens developed in
the crude cultures from Belize, together with more intermediate
sized cells (Fig. 34H-J). These specimens were otherwise characteristic for C. roenbergensis. It is typical of some heterotrophs
that unusually large or pseudopodia-rich “monster cells” develop in dense cultures (e.g. Actinomonas, unpubl obs.). Large
cells (up to 9 pm) were also observed in dense cultures by Larsen
& Patterson [27], and these were regarded as “eco-morphs” of
C. roenbergensis.
Previous records (marine). Tropical Australia [27]; the Baltic
Sea [ 14, 72, 731; Antarctica and Greenland [7 11.
CHROMULINALES Pascher 19 12,
sensu Preisig et al. [50] (ICBN)
(= CHROMOMONADINA Klebs, 1893)
Paraphysomonas De Saedeleer 1929
Biflagellate, heterokont flagellates, possessing a cell body covered with siliceous scales [50].
See Takahashi [59] for detailed descriptions of the species
below. Tables 3 & 4 summarize dimensions of cells and scales
measured from EM whole mounts (Paraphysomonasantarctica:
16 different cells; Paraphysomonas imperforata: three different
cells).
Paraphysomonas antarctica Takahashi 1987
(Fig. 18-2 1, 34L, Tables 3, 4)
Remarks. This species is a member of the “imperforata complex.” The scales resemble those of P. imperforata sensu stricto,
but according to Takahashi [59] they differ in three respects: 1)
the diameter of the basal plate and the length of the spines are
twice as great as those of P. imperforata; 2) the ratio of the length
0)
of the spine tip to the length of the total spine is ‘4.6 (‘4.6
compared to V 2 (Y2
1) in P. imperforata; 3) the thickness of
the spine at the transition zone between the tip and the main
part of the spine changes abruptly, instead of gradually as in P.
imperforata. These distinctions are only valid on the condition
that the large-scaled forms of P. imperforata sensu lato are transferred to other species [59].
The smallest scales of the cells of P. antarctica from Belize
(Table 3) are generally much smaller than previously reported
[59]. This invalidates Takahashi’s distinction No. 1. The finding
of intermediate scale types, which fit the size range given for P.
imperforata sensu stricto, but that have spines with an abrupt
change in thickness, a characteristic for P. antarctica (Fig. 22,
Table 4), invalidates Takahashi’s distinction No. 3. The dimensions of the scales of the specimens from Belize may cover
both P. antarctica and P. imperforata sensu stricto (Table 3).
This “polymorphy” may be exhibited by the scales of a single
cell (Fig. 18-2 1, Table 4). All scales may, however, be referred
to P. antarctica strictly following Takahashi’s distinction No.
2. At present, this distinction is the only criterium on which P.
antarctica may be separated.
Paraphysomonas antarctica has previously been reported only
from cold seawater and sea-ice, and the scales of the cold-water
specimens were found to be in close agreement with the description [7 1, 721. It is interesting to note that the present aberrant specimens of P. antarctica and P. imperforata originate
from warm-water cultures (20” C), indicating that the temperature may influence the size of the scales.
Previous records (marine). Known from temperate, arctic
and antarctic sites [72].
-+
+
Paraphysomonas imperforata Lucas 1967
(Fig. 22-24, Tables 3-4)
Remarks. Paraphysomonas imperforata is a well studied and
extremely polymorphic species [49, 591. The size values of the
284
J. EUK. MICROBIOL., VOL. 40, NO. 3, MAY-JUNE 1993
specimens from Belize have been included for comparison with
P. antarctica from the same locality.
Previous records (P.imperforata sensu lato). Widespread in
fresh and marine waters at arctic, antarctic, temperate, and
sub-tropical sites [59].
INCERTAE SEDIS TAXA (ICZN)
Lufisphaera Belcher & Swale, 1975
Species of this little known genus are globular or elliptical
cells covered with two types of hollow meshwork scales: spine
scales and base scales. The cells range 1.5-3.5 pm in diameter
and the spine scales range 0.1-0.4 pm in width and 1.8-2.9 pm
in length. The genus is also characterized by tubulocristate mitochondria, and a lack of flagella and pseudopodia [3]. Belcher
& Swale [3] described four species distinguished by scale morphology.
Species of Lufiphaera are likely to be overlooked because of
their small size. The genus has not been reported from fresh
water since the description [3] and has been encountered a few
times in seawater [3, 63, 72, 731.
The scale-covered organisms encountered in the Belizean
samples superficially resemble named species of Luffisphaera in
both morphology and size. However, the scale morphology of
these organisms is significantly different and they are described
as three new species. Figure 33 show drawings ofthe spine scales
of the new species, Table 5 summarizes scale dimensions, measured from five (Lufiphaera bulbochaete; Lufiphaera turriformis) or three (Lufiphaera longihastis) different cells.
Lumphaera bulbochaete Vms n. sp.
(Fig. 25-26, 33A, Table 5)
(The epithet refers to Bufbochaete, a green alga that cames
bristles with bulbous bases.)
Diagnosis. Spine scales consist of a cylindrical base supporting a dome-shaped part, which gradually tapers to a narrow
tip. This tip is mounted by a thin spine. The lattice of the dome
is composed of regular hexagonal perforations.
Type specimen. Collected at Carrie Bow Cay pier, Belize (=
specimen in Fig. 25).
Description. Cell spherical, 3 4 pm in diameter (dried cells)
covered with a dense layer of scales. Most of these are base
scales, while 20-40 are spine scales. Spine scales consist of a
cylindrical base supporting a dome-shaped part, which gradually
tapers to a narrow tip. This tip is mounted by a thin spine. The
base comprises two differently patterned parts: a proximal lattice
with an irregular pattern, and a distal lattice with a regular
pattern of square perforations. The lattice of the dome is composed of regular hexagonal perforations. The tip of the dome
and the spine consist of smooth, homogenous, non-lattice material. The spine scales are 1.5-2.2 pm in total length.
Lumphaera longihastis Vers n. sp.
(Fig. 27-29, 33C, Table 5 )
(“Longihastis” is Latin for “long-speared.”)
Diagnosis. Spine scales consist of a cylindrical base supporting a cone-shaped part, which gradually tapers to a narrow
tip. This tip is mounted by a long, thin whip-like spine. The
base and the cone are each composed of a proximal lattice with
criss-cross pattern and a distal lattice with longitudinal rods
only.
Type specimen. Collected at Carrie Bow Cay pier, Belize (=
specimen in Fig 27).
Description. Cell spherical or almost so, 3 4 . 2 pm in di-
ameter (dried cells), covered with a dense layer of scales. Most
of these are base scales, but more than 20 or so are furnished
with spines. Spine scales consist of a cylindrical base supporting
a cone-shaped part, which gradually tapers to a narrow tip. This
tip is mounted by a long, thin whip-like spine. The base and
the cone are both composed of two differently patterned parts:
a proximal lattice with criss-cross pattern and a distal lattice
with longitudinal rods only. The top of the cone and the spine
consist of smooth, homogenous, non-lattice material. The spine
scales are 2 . 5 4 pm in total length.
Lumphaera turriformis V0rs n. sp.
(Fig. 30-32, 33B, Table 5)
(“Tumformis” is Latin for “tower-shaped.”)
Diagnosis. Spine scales consist of a cylindrical base supporting a cone-shaped part, which gradually tapers to a long
narrow rod. This rod is mounted by a long, thin rod of about
half its width. The lattice of the cone is composed of distinct
longitudinal ribs, between which is a lattice of small square or
hexagonal perforations.
Type specimen. Collected at Carrie Bow Cay pier, Belize (=
specimen in Fig. 30).
Description. Cell spherical, 4-5 pm in diameter, covered with
a dense layer of scales. Most of these are base scales, but more
than 50 or so are spine scales. Spine scales consist o f a cylindrical
base supporting a cone-shaped part, which gradually tapers to
a long narrow rod. This rod is mounted by a long, thin rod of
about half its width. The base comprises two differently patterned parts: a proximal lattice with irregular pattern, and a
distal lattice with a regular pattern of square perforations. The
lattice of the cone is composed of distinct longitudinal ribs.
Between these lies a lattice of one or more rows of square or
hexagonal perforations. The distal rods consist of smooth or
finely striated non-lattice material. The spine scales are 2.9-3.8
Wm in total length.
DISCUSSION
The study of non-ciliate, heterotrophic protists from Belize
and Tenerife was based on a small number of crude cultures
and water samples, but nevertheless revealed a highly diverse
community of heterotrophs. The sample from Tenerife is considered too small (1 liter) to justify a comparison between the
records from the two sites, whereas the combined species list
of marine heterotrophic protists from both Belize and Tenerife
may be compared with that of Arctic (Canada and Greenland
[7 I]) and temperate (the Baltic [72, 731) regions (Table 1). One
of the most interesting results is the similarity between these
species lists. The list from the tropics contains 34 taxa, of which
13 are shared with the list from the Arctic and 25 taxa are in
common with the list from the temperate Baltic. The difference
in numbers may be because of the small number of samples of
arctic protists that have been studied, while the Baltic protists
were surveyed in more detail over a three-year period. Fifteen
species encountered during the present study also have been
found in tropical sediments [27]. Twelve taxa were encountered
in all regions (Diaphanoeca grandis, Bodo designis, Bod0 saliens,
Amastigomonas debruynei, Ancyromonas sigmoides, Telonema
subtile, Cafeteria roenbergensis, Paraphysomonas antarctica,
Paraphysomonas imperforata, Actinomonas mirabilislPteridomonas danica, Heterophrys myriapoda, Vanella aberdonica)
(Table 1).
The majority of widely distributed taxa reported here are
bacterivorous flagellates. It has been speculated that small heterotrophs like these may easily attain a widespread or cosmo-
V0RS-(SUB)TROPICAL HETEROTROPHIC PROTISTS
politan distribution because of the numerous means of dispersal
for small organisms [36], and because their survival and potential for rapid growth are facilitated by the ubiquity of their prey
[13, 72, 751.
The present study also revealed a bacterivorous species with
an apparently restricted distribution, the salpingoecid choanoflagellate S. camelopardula. This species has previously been
recorded only from California (temperature: 19-25' C, salinity:
30-339~S) [37] and from the Red Sea (temperature: 15-25" C,
salinity: ca. 40% S) [61]. In the present study, temperature and
salinity was about 26" C and 3 4 9 S
~ respectively. Salpingoeca
camelopardula may thus be an oceanic warm-water endemic,
explaining its apparent absence in the Baltic (salinity usually
below 309m S) [62, 69, 72, 731 and at arctic localities (temperature usually below 13°C) [63,67-69,7 I]. However, the absence
of the species in these areas may simply signify that sampling
has been insufficient [ 131.
Limited distributions are known from the more intensively
studied acanthoecid choanoflagellates. Thomsen [63] and
Thomsen et al. [66] reported nine cosmopolitan species of loricate choanoflagellates (Bicosta minor, Bicosta spinlfera, Calliacantha natans, Calliacantha simplex, Cosmoeca norvegica,
Cosmoeca ventricosa, Parvicorbicula circularis, Parvicorbicula
socialis, Pleurasiga minima); eight species restricted to warm
waters (Aphaloecion pentacanthum, Calotheca alata, Campyloacantha spinifera, Cosmoeca ceratophora, Cosmoeca phuketensis, Platypleura acuta, Platypieura perforata, Stephanacantha
campaniformis); and five species confined to colder waters (Calliacantha longicaudata, Conion groenlandicurn, Pleurasiga caudata, Parvicorbicula quadricostata, Parvicorbicula serratula). A
large number of species are widespread, but not truly cosmopolitan [63, 661.
In conclusion, the studies of the diversity of non-ciliate freeliving heterotrophic protists conducted so far [27, 63, 66, 7 173 and references therein] give evidence that certain groups of
flagellates are geographically widespread (cryptophyceans, kinetoplastids, bicosoecids, chromulinids, pedinellids and apusomonads), while others have limited distributions (some
choanoflagellates). The available information is, however, still
too scarce for more detailed biogeographical speculations.
Whether the ubiquitous species are truly eurytherm and euryhaline, or whether they have optimal growth in a limited range
of temperatures or salinities, have yet to be addressed. Nor is
it known whether we are dealing with an insufficient resolution
at the species level, so that genetically different species with
different ecological preferences may not be recognized because
they are morphologically identical [ 131. Species complex of such
"sibling species" are known for the ciliates [ 131. This is a field
of research in need of more attention, preferably in the form of
more extensive studies of the diversity of heterotrophs in polar
and tropical waters and of the abundance and autecology of
individual species.
ACKNOWLEDGMENTS
Jacob Larsen, 0Jvind Moestrup, Kristian M. Christensen and
Susan Rasmussen are thanked for collecting the material. Thanks
are also due to Tyge Christensen for guidance in nomenclatural
matters and to David J. Patterson, 0jvind Moestrup, Helge A.
Thomsen and Maria A. Faust for criticism and suggestions.
Lisbeth T. Haukrogh, Lene Christiansen and Karin G. Jensen
are thanked for technical assistance.
The author acknowledges financial support of the Danish National Agency for Environmental Protection (Hav90), the Carlsberg Foundation, and the University of Copenhagen. Financial
support from the Smithsonian Institution's Caribbean Coral
285
Reef Ecosystem Program for collecting specimens in Belize is
also gratefully acknowledged. This paper is contribution NO.
340 from the CCRE.
LITERATURE CITED
1. Ariztia, E. V., Andersen, R. A. & Sogm, M. L.
1991. A new
phylogeny for chromophyte algae using 16s-like rRNA sequences from
Mallomonas papillosa (Synurophyceae) and Tribonema aequale (Xanthophyceae). J. Phycol., 27:428436.
2. Bachmann, H. 191 1. Das phytoplankton des Siisswassers mit
besonderer Beriicksichtigung des Vienvaldsdttersees. Mitt. der Naturf:
Ges. Luzern, VI: 1-5 1.
3. Belcher, J. H. & Swale, E. M. F. 1975. Lumphaera gen. nov.,
an enigmatic scaly micro-organism. Proc. R. SOC.Lond. B, 188:495499.
4. Boucaud-Camou, E. 1966. Les choanoflagellts des cotes de la
Manche: I. systtmatique. Bull. SOC.Linn. Normandie (10. ser.), 7: I9 1209.
5. Bovee, E. C. 1985. Class Filosea Leidy, 1879. In: Lee, J. J.,
Hutner, S. H. & Bovee, E C. (ed.), An illustrated Guide to the Protozoa.
Society of Protozoologists, Lawrence, KS. Pp. 228-246.
6. Burck, C. 1909. Studien iiber einige Choanoflagellaten. Arch.
Protistenk., 16:169- 186.
7. Caron, D. A. & Swanberg, N. R. 1990. The Ecology ofplanktonic
Sarcodines. Aquatic Sci., 3: 147-180.
8. Davis, P. G., Caron, D. A. & Sieburth, J. M. 1978. Oceanic
amoebae from the North Atlantic: culture, distribution,and taxonomy.
Trans. Amer. Microsc. SOC.,97:73-88.
9. De Saedeleer, H. 1927. Notes de Protistologie I. Crasptdomonadines: mattriel systkmatique. Ann. SOC.Roy. Zool. Belgique, LVIII:
117-147.
10. Dunkerly, J. S. 1910. Notes on the Choanoflagellate genera
Salpingoeca and Polyoeca, with description of Polyoeca dumosa sp. n.
Ann. Mag. Nut. Hist. (8 ser.), 5:186-191.
1 1. Ellis, W. N. 1930. Recent researches on the Choanoflagellata
(Craspedomonadines)(freshwater and marine) with description of new
genera and species. Ann. SOC.Roy. Zool. Belgique, 60:49-88.
12. Ettl, H. 1970. Ein Beitrag zur Kenntnis der Algenflora Tirols
11. Ber. Nut.-Med. Ver. Innsbruck, 58:89-124.
13. Fenchel, T. 1987. Ecology of Protozoa. The Biology of Freeliving Phagotrophic Protists. ScienceTech Publishers, Madison, WI and
Springer-Verlag, Berlin.
14. Fenchel, T. & Patterson, D. J. 1988. Cafeteria roenbergensis
nov. gen., nov. sp., a heterotrophic microflagellate from marine plankton. Mar. Microb. Food Webs, 3:9-19.
15. Franct, R. 1897. Der Organismus der Craspedomonaden.Koniglische Ungarische Natunvissenschaftliche Gesellschaft, Budapest.
16. Gajadhar, A. A,, Marquardt, W. C., Hall, R., Gunderson, J.,
Ariztia-Carmona, E. V. & Sogin, M. L. 1991. Evolutionary relationships among apicomplexans, dinoflagellates, and ciliates: Ribosomal
RNA sequences of Sarcocystis muris, Theileria annulata and Ctypthecodinium cohhnii. Mol. Biochem. Parasitol. 45: 147-1 54.
17. Greuter, W. (ed.) 1988. International code of botanical nomenclature. Regnum Vegetabile, 118:1-328.
18. Hansen, P. J. 1991. Quantitative importance and trophic role
of heterotrophic dinoflagellates in a coastal pelagial food web. Mar.
Ecol. Progr. Ser.. 73:253-261.
19. Hara, S., Tanoue, E., Zenimoto, M., Komaki, Y. & Takahashi,
E. 1986. Morphology and distribution of heterotrophic protists along
75" E in the Southern Ocean. Mem. Nat. Inst. Polar Res., Spec. Issue,
40~69-80.
20. Hill, D. R. A. 1991. The diversity of cryptomonads. In: Patterson, D. J. & Larsen, J. (ed.), Biology of Free-living Heterotrophic
Flagellates. Syst. Assoc. Spec. Vol., 45. Clarendon Press, Oxford. Pp.
235-240.
21. James-Clark, H. 1867. On the Spongiae Ciliatae as Infusoria
Flagellata. Mem. Boston SOC.Nut. Hist., I(3):305-342.
22. Johnson, P. W., Hargraves, P. E. & Sieburth, J. M. 1988. U1trastructure and ecology of Calycomonas ovalis Wulff 1919, (Chrysophyceae) and its redescription as a testate rhizopod, Paulinella ovalis n.
comb. (Filosea, Euglyphina). J. Protozool., 356 18426.
286
J. EUK. MICROBIOL.. VOL. 40, NO. 3, MAY-JUNE 1993
23. Kent, W. S. 1880 (1880-1882). A Manual ofthe Infusoria, Vol.
1-3. Bogue, London.
24. Kies, L. 1974. Elektronenmikroskopische Untersuchungen an
Paulinella chromatophora Lauterborn, einer Thekamobe mit blaugriinen Endosymbionten (Cyanellen). Protoplasma, 80:69-89.
25. Klug, G. 1936. Salpingorhizagen. nov., eineneuecraspedomonadaceen-Gattung. Arch. Protistenk., 87:117-122.
26. Kugrens, P. & Lee, R. E. 1991. Organization of cryptomonads.
In: Patterson, D. J. & Larsen, J. (ed.), Biology of Free-living Heterotrophic Flagellates. Syst. Assoc. Spec. Vol., 45. Clarendon Press, Oxford.
Pp. 219-233.
27. Larsen, J. & Patterson, D. J. 1990. Some flagellates (Protists)
from tropical marine sediments. J. Nut. Hist., 2430 1-937.
28. Lauterborn, R. 1895. Protozoenstudien. 11. Paulinella chromotophora nov. gen. nov. spec., ein beschalter Rhizopode des Susswassers
mit blaugriinen chromatophorenartigen Einschliissen. Zeitschr. Wissensch. Zool., 59537-544
Taf. XXX.
29. Leadbeater, B. S. C. 1977. Observations on the life-history and
ultrastructure of the marine choanoflagellate Choanoeca perplexa Ellis.
J. Mar. Biol. Ass. U.K.. 57:285-301.
30. Leadbeater, B. S. C. 1991. Choanoflagellate organization with
special reference to loricate taxa. In: Patterson, D. J. & Larsen, J. (ed.),
Biology of Free-living Heterotrophic Flagellates. Syst. Assoc. Spec. Vol..
45. Clarendon Press, Oxford. Pp. 241-258.
3 1. Leidy, J. 1879. Freshwater rhizopods of North America. Report
U. S. Geol. Surv. Terr., 12:l-325.
32. Lemmermann, E. 1914. vagellatae I. In: Pascher, A. (ed.), Die
Siisswasser-Flora Deutschlands, Ostereichs und der Schweiz. Gustav
Fischer, Jena. Vol. 1.
33. Mignot, J. P. 1974. Etude ultrastructurale des Bicoeca, protiste
flagell&. Protistologica., 10:5 4 3-5 6 5.
34. Moestrup, 0. 1984. Further studies on Nephroselmis and its
allies (Prasinophyceae) 11. Mamiella gen. nov., Mamiellaceae fam. nov.,
Mamiellales ord. nov. Nord. J. Bot. 4: 109-1 2 1.
35. Moestrup, 0.& Thomsen, H. A. 1980. Preparations of shadow
cast whole mounts. In: Gantt, E. (ed.), Handbook of Phycological Methods. Developmental and Cytological Methods. Cambridge Univ. Press.
Pp. 385-390.
36. Noland, L. E. & Gojdics, M. 1967. Ecology of free-living protozoa. In: Tze-Tuan, C. (ed.), Research in Protozoology, Pergamon
Press, Oxford. 2:2 15-266.
37. Noms, R. E. 1965. Neustonic marine Craspedomonadales
(Choanoflagellates)from Washington and California. J. Protozool., 12:
589602.
38. Ogden, C. G. & Hedley, R. H. 1980. An Atlas of Freshwater
testate Amoebae. Oxford University Press.
39. Page, F. C. 1987. The classification of “naked” amoebae (Phylum Rhizopoda). Arch. Protistenk., 133:199-2 17.
40. Page, F. C. 1991. Nackte Rhizopoda. In: Page, F. C. & Siemensma, F. J., Nackte Rhizopoda und Heliozoea. Protozoenfauna Band
2. Gustav Fisher Verlag, Stuttgart. Pp. 1-1 70.
41. Patterson, D. J. 1983. On the organization of the naked filose
amoeba, Nuclearia moebiusi Frenzel, I897 (Sarcodina, Filosea) and its
implications. J. Protozool., 30:301-307.
42. Patterson, D. J. 1989. The evolution of protozoa. Mem. Inst.
Oswaldo Cmz. Rio de Janeiro. Suppl. I., 83:580-600.
43. Patterson, D. J. 1989. Stramenopiles: chromophytes from a
protistan perspective. In: Leadbeater, B. S. C. & Diver, W. L. (ed.),
Chromophyte Algae: Problems and Perspectives. Clarendon Press, Oxford. Pp. 357-379.
44. Patterson, D. J. & Delvinquier, B. L.J. 1990. The fine structure
of the cortex of the protist Protoopalina australis (Slopalinida, Opalinidae) from Litoria nasuta and Litoria inermis (Amphibia: Anura: Hylidae) in Queensland, Australia. J. Protozool., 37:449455.
45. Patterson, D. J. & Larsen, J. 1991. (ed.) Biology of Free-living
Heterotrophic Flagellates. Syst. Assoc. Spec. Vol., 45. Clarendon Press,
Oxford.
46. Patterson, D. J. & Larsen, J. 1991. Nomenclatural problems
with protists. In: Hawksworth, D. L. (ed.), Improving the Stability of
Names: Needs and Options. Regnum Vegetabile, 123: 197-208.
47. Patterson, D. J. & Larsen, J. 1992. A perspective on protistan
nomenclature. J. Protozool., 39: 125-1 3 1.
+
48. Patterson, D. J. & Zolffel, M. 1991. Heterotrophic flagellates
of uncertain taxonomic position. In: Patterson, D. J. & Larsen, J. (ed.),
Biology of Free-living Heterotrophic Flagellates. Syst. Assoc. Spec. Vol.,
45. Clarendon Press, Oxford. Pp. 427-476.
49. Preisig, H. R. & Hibberd, D. J. 1982. Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera I. Nord.
J. Bot., 2:397420.
50. Preisig, H. R., Vers, N. & Hallfors, G. 1991. Diversity ofheterokont heterotrophic flagellates. In: Patterson, D. J. & Larsen, J. (ed.),
Biology of Free-living Heterotrophic Flagellates. Syst. Assoc. Spec. Vol.,
45. Clarendon Press, Oxford. Pp. 36 1-400.
51. Ride, W. D. L., Sabrosky, C. W., Bernardi, C. W. & Melville, R.
V. (ed.) 1985. The International Commission on Zoological Nomenclature: International Code of Zoological Nomenclature, 3rd ed. International Trust for Zoological Nomenclature in Association with British
Museum London, and University of California Press, Berkeley and Los
Angeles.
52. Ruinen, J. 1938. Notizen iiber Salzflagellaten. 11. Uber die Verbreitung der Salzflagellaten. Arch. Protistenk., 90:2 10-258.
53. Schlegel, M. 1991. Protist evolution and phylogeny as discerned
from small subunit ribosomal RNA sequence comparisons. Europ. J.
Protistol., 27:207-219.
54. Siemensma, F. J. 1991. Heliozoea. In: Page, F. C. & Siemensma, F. J., Nackte Rhizopoda und Heliozoea. Protozoenfauna Band 2.
Gustav Fisher Verlag, Stuttgart. Pp. 171-290.
5 5 . Skuja, H. 1939. Beitrag zur Algenflora Lettlands 11. Acta Horti
Botanici Universitatis latviensis, 11/12:4 1-169.
56. Skuja, H. 1956. Taxonomische und biologische Studien das
Phytoplankton Schwedischer Binnengewasser. Nova Acta Regiae SOC.
Scient. Uppsaliensis. 4th ser., 16: 1 4 0 3 .
57. Stein, F. R. 1878. Der Organismus der Infusionstiere. 111. Abteilung. I. H2lfte. Leipzig.
58. Stokes, A. C. 1886. New members ofthe infusorialorder ChoanoFlagellata. S.K.-IV. Amer. Monthl. Microsc. J., 7:227-229.
59. Takahashi, E. 1987. Loricate and scale-bearing protists from
Liitzow-Holm Bay, Antarctica 11. Four marine species of Paraphysomonus (Chrysophyceae)including two new speciesfrom the fast-ice covered
area. Jap. J. Phycol. (S6rui). 35:155-166.
60. Thomsen, H. A. 1977. External morphology of the choanoflagellate Salpingoeca gracilis James-Clark. J. Mar. Biol. Assoc. U.K.. 57:
629634.
61. Thomsen, H. A. 1978. Nanoplankton from the Gulf of Elat (=
Gulf of Aqaba), with particular emphasis on choanoflagellates.Israel J.
Zool.. 27:3444.
62. Thomsen, H. A. 1979. Electron microscopical observations on
brackish-water nannoplankton from the Tvarminne area, SW coast of
Finland. Acta. Bot. Fennica. 11O:ll-37.
63. Thomsen, H. A. 1981. Planktonic choanoflagellatesfrom Disko
Bugt, West Greenland with a survey of the marine nanoplankton of the
area. Meddr. Crml. Biosci.. 8:3-35.
64. Thomsen, H. A. &Buck, K. R. 1991. Choanoflagellatediversity
with particular emphasis on the Acanthocidae. In: Patterson, D. J. &
Larsen, J. (eds), Biology of Free-living Heterotrophic Flagellates. Syst.
Assoc. Spec. Vol., 45. Clarendon Press, Oxford. Pp. 259-284.
65. Thomsen, H. A., Buck, K. R., Bolt, P. A. & Gamson, D. L.
1991. Fine structure and biology of Cryothecomonas gen. nov. (Protista
incertae sedis) from the ice biota. Can. J. Zool., 69:1048-1070.
66. Thomsen, H. A., Buck, K. R. & Chavez, F. P. 1991. Choanoflagellates of the central California waters: taxonomy, morphology and
species assemblages. Ophelia, 33: 131-1 64.
67. Throndsen, J. 1969. Flagellates of Norwegian coastal waters.
Nytt Mag. Bot.. 16:161-216.
68. Throndsen, J. 1970. Flagellates from Arctic waters. Nytt Mag.
Bot., 17:49-57.
69. Throndsen, J. 1974. Planktonic choanoflagellates from North
Atlantic waters. Sarsia, 56:95-122.
70. Turley, C. M. & Carstens, M. 199I . Pressure tolerance of oceanic flagellates: implications for remineralization of organic matter. DeepSea Res.. 38:403-413.
7 1. Vers, N. 1993. Heterotrophic amoebae, flagellatesand heliozoa
from arctic marine waters (N.W.T. Canada and West Greenland). Polar
Biol., 13:113-1 26.
V0RS- (SUB)TROPICAL HETEROTROPHIC PROTISTS
72. Vers, N. 1992. Heterotrophic amoebae, flagellates and heliozoa
from the Tvarminne area, Gulf of Finland, in 1988-1990. Ophelia,
36(1):1-109.
73. Vers, N. 1992. Heterotrophic protists (excl. dinoflagellates, loricate choanoflagellates and ciliates). In: Thomsen, H. A. (ed.), Plankton
from Inner Danish Waters. An Analysis of the Autotrophic and Heterotrophic Plankton in Kattegat. Hav90 Report. Danish National Agency for Environmental Protection, 11:195-249. (in Danish, with English
summary, species lists and 130 figures.)
74. Vars, N. 1992. Ultrastructure and autecology of the marine
heterotrophic flagellate Leucocryptos marina (Braarud) Butcher 1967
287
(Katablepharidaceae/Kathablepharidae),with a discussion of the genera
Leucocryptosand Katablepharis/Kathablepharis.Europ. J. Protistol.. 28:
369-389.
75. Vers N., Havskum H. & Johansen, B. 1990. Electron microscopical observations on some species of Paraphysornonas (Chrysophyceae) from Danish lakes and ponds. Nova Hedwigia, 50:337-354.
76. Zhukov, B. F. & Karpov, S. A. 1985. Freshwater Choanoflagellates. Acad. Sci. USSR, Inst. Biol. Inland Waters, Leningrad.
Received 5-18-92, 9-2-92, 11-24-92; accepted 11-30-92
J. Euk. Microbiol., 40(3), 1993,pp. 287-297
0 1993 by the Society of Protozoologists
Gametogenesis and Sporogony of Hepatozoon mocassini
(Apicomplexa: Adeleina: Hepatozoidae) in an Experimental
Mosquito Host, Aedes mopti
AMY LOWICHIK,'.* H. NORBERT LANNERS,** ROBERT C. LOWRIE, JR.,** and NORWOOD E. MEINERS***
*Department of Tropical Medicine and Parasitology, Tulane University,New Orleans, Louisiana 701IS, and
**Departments of Parasitology and ***Pathology,Tulane Regional Primate Research Center, Covington, Louisiana 70433
ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti, an experimentalmosquito
host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after
mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding,
gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within
the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual
differentiation were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes
as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a parasitophorous
vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporozoites developing within sporocysts
appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containingapproximately 16 sporozoiteswere observed
upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endoplasmic reticulum were located anterior and
posterior to the sporozoite nucleus. Free sporozoites were not observed.
Supplementary key words. Haemogregarine, snake parasites, ultrastructure.
T
HE family Hepatozoidae Wenyon, 1926 is characterized by
merogony occumng in vertebrate tissues and by gamonts
located within circulating blood cells of the vertebrate host.
Adeleid-type syzygy within the invertebrate host is followed by
development of oocysts that contain sporocysts in which sporozoites are found [46]. Miller created the genus Hepatozoon to
accommodate H . perniciosurn, a haemogregarine that infects
leukocytes of white rats [25]. Gamonts of H. perniciosurn pair
up in syzygy, undergo sexual differentiation, and fuse in the gut
lumen of a mite; the resulting zygote migrates between gut epithelial cells into the haemocoele. A variety of mammalian and
reptilian haemogregarines have since been assigned to this genus
after mature oocysts containing sporocysts were observed in
natural or experimental invertebrate hosts. Detailed studies of
the events in sexual reproduction of Hepatozoon spp. are few,
and variations observed may be of taxonomic significance [2,
5,421. In the present study, we report the ultrastructural features
of gametogenesis and sporogony of another reptile haemogregarine parasite, Hepatozoon rnocassini (Laveran, 1902).
I To whom correspondence should be addressed. Present address:
Department of Pathology, University of Texas Southwestern Medical
School, Dallas, Texas, 75235-9072.
MATERIALS AND METHODS
Collection of snakes and examination of blood. Cottonmouths (Agkistrodon piscivorus leucostorna (Troost)), banded
water snakes (Nerodiafasczata confluens (Blanchard)), diamondbacked water snakes (Nerodia rhornbifera rhornbifera (Hallowell)) and canebrake rattlesnakes (Crota1u.s horridus atricaudatus
Latreille) naturally infected with haemogregarine parasites were
collected from various locations outside New Orleans and
LaPlace in southeastern Louisiana. Blood for thin smears was
obtained by cutting the tips of the snake tails with a razor blade.
Air-dried blood smears, fixed in absolute methanol for five min,
were stained for 1 h with 3% (v/v) Giemsa's solution (Ricca
Chemical Co., Arlington, TX) in phosphate-buffered water, pH
7.2, and examined at 400 x magnification.
Feeding of mosquitoes. Laboratory-reared Aedes aegypti,
black-eye Liverpool strain, were raised from eggs, and the adult
mosquitoes were maintained in pint cardboard cartons at 27"
C in a Precision Low Temperature Incubator, Model 8 18 (Precision Scientific Group, G. C. A. Corp., Chicago, IL). Nutrition
and humidity were provided by cotton pads moistened with
10% Karo syrup solution (Best Foods, C. P. C. International
Inc., Englewood Cliffs, NJ), supplemented with 0.00 1% (v/v)
nordihydroguaiaretic acid (Sigma Chemical Co., St. Louis, MO)