Nebela kivuense Gauthier-Lievre et Thomas, 1961 (Amoebozoa, Arcellinida), Missing for a Half-century; Found 11,500 km from "home"

Abstract

In 1961, the testate amoeba Nebela kivuense Gauthier-Lièvre et Thomas, 1961 was described for the first and only time from an area near Lake Edward in the Democratic Republic of the Congo (at 0.002° N Latitude). The lack of recent reports of this species, despite exhaustive surveys of the testate amoebae fauna of the major continents of the world, suggested that N. kivuense was a rare species perhaps endemic to a small, local equatorial region of the African continent. This paper reports its rediscovery from two wetland-conifer forest ecosystems in southern Ontario, Canada (at 44° N Latitude), thus changing dramatically our previous perception of its very restricted global distribution. This has implications for the idea held by many students of biogeography that there is a special category of microscopic protists that contains truly rare species and their rarity, perhaps together with specific habitat requirements and tolerances, limits opportunities for dispersal around the world. The N. kivuense story is a clear example of the dangers of inferring endemism from rarity.

Full text

Acta Protozool. (2015) 54: 283–288
www.ejournals.eu/Acta-Protozoologica
doi:10.4467/16890027AP.15.023.3537
ActA
Protozoologica
Nebela kivuense Gauthier-Lièvre et Thomas, 1961 (Amoebozoa,
Arcellinida), Missing for a Half-century; Found 11,500 km from “home”
Kenneth H. NICHOLLS
S-15 Concession 1, RR #1, Sunderland, Ontario, Canada
Abstract. In 1961, the testate amoeba Nebela kivuense Gauthier-Lièvre et Thomas, 1961 was described for the rst and only time from an
area near Lake Edward in the Democratic Republic of the Congo (at 0.002° N Latitude). The lack of recent reports of this species, despite
exhaustive surveys of the testate amoebae fauna of the major continents of the world, suggested that N. kivuense was a rare species perhaps
endemic to a small, local equatorial region of the African continent. This paper reports its rediscovery from two wetland-conifer forest eco-
systems in southern Ontario, Canada (at 44° N Latitude), thus changing dramatically our previous perception of its very restricted global
distribution. This has implications for the idea held by many students of biogeography that there is a special category of microscopic protists
that contains truly rare species and their rarity, perhaps together with specic habitat requirements and tolerances, limits opportunities for
dispersal around the world. The N. kivuense story is a clear example of the dangers of inferring endemism from rarity.
Key words: Nebela, testate amoeba, Hyalospheniidae, protist biogeography.
Address for correspondence: Kenneth H. Nicholls, S-15 Conces-
sion 1, RR #1, Sunderland, Ontario, Canada L0C 1H0; E-mail:
khnicholls@interhop.net
INTRODUCTION
The testate amoeboid genus Nebela is one of sev-
eral moss and soil-dwelling genera recently assigned to
the Hyalospheniidae (Amoebozoa, Arcellinida) by Ko-
sakyan et al. (2012, 2013). Nebela species are charac-
terized by a hyaline organic test to which are cemented
various siliceous scales of other protists, notably those
of the genera Euglypha, Trinema, Corythion, and oth-
ers. These are scavenged from the environment from
dead cells, or may be actively ingested during preda-
tory feeding on living prey, the scales of which are then
secreted to the exterior of the Nebela test (Meisterfeld
2002). Nebela tests are usually attened disc-shaped or
elongated bulb-like structures possessing an elliptical
pseudostome with a smooth thickened rim.
Among the very smallest Nebela species is N. ki-
vuense, the only veried record of which is from a sin-
gle location (the type locality) in East-Central Africa
(Gauthier-Lièvre and Thomas 1961). This taxon also
appeared among a listing of 22 Nebela species from
Romania (Godeanu 1972). That report remains unsub-
stantiated, however, owing to the lack of any accom-
panying descriptive material (measurements, drawings,
images, etc.) that would support its identication. The
purpose of this note is to report the occurrence of N. ki-

K. H. Nicholls
284
vuense from Ontario, Canada, because it has important
implications for the theories of biogeography of small
rare protists.
MATERIALS AND METHODS
Samples of moss from shaded, wet fen habitats in south-central
Ontario were collected in April and May of 2000 and July of 2001
by picking several whole plants, including the older brown ground-
level portions, and rinsing vigorously with distilled water in 500
mL plastic bottles to dislodge testate amoebae attached to stems and
leaves. Several such samples were collected within plots of approxi-
mately 100 m in diameter at two locations in separate watersheds.
These were located at: 1) 44°08′30″N, 79°06″W (Uxbridge Brook
of the Pefferlaw River watershed), and 2) 44°10′40″N, 79°07′30″W
(Beaver River watershed). At both locations, the arboreal oversto-
ry was dominated by White Spruce (Picea glauca), Black Spruce
(Picea mariana), Balsam Fir (Abies balsamea) and Eastern White
Cedar (Thuja occidentalis). Samples containing Nebela kivuense
were associated with the mosses Hylocomium splendens (Hedw.)
B.S.G., Sphagnum wulanum Girgensohn, and two other unidenti-
ed Sphagnum species.
Laboratory methods have been described elsewhere (e.g., Nich-
olls, 2007). Measures of central tendencies of test morphology were
summarized using the descriptive statistical methods described in
Costat (Cohort Software 1995). Owing to some similarity in the
shape of the tests of Nebela kivuense and Nebela militaris, which
co-occurred in some of the same samples, measurements of speci-
mens of both taxa were made and compared statistically.
RESULTS AND DISCUSSION
The sizes and shapes of test of N. kivuense in the
Ontario collections generally agreed well with those
described and illustrated (Fig. 1) by Gauthier-Lièvre
and Thomas (1961). Ontario specimens were more var-
iable in the degree to which they were slightly curved in
plan view (Figs 2–6, 12a, 13a, 14), and in the degree of
taper from the widest region near the aboral end to the
anterior in both plan (Figs 2–6, 12a, 13a, 14) and lateral
(Figs 7–11, 12b, 13b) views. In all tests examined, the
pseudostomal aperture was outlined by a thickened rim
of organic cement (Fig. 15). A cyst plate was found in
some specimens constructed about 15–20 µm from the
anterior pseudostomal aperture and spanning the entire
width of the interior of the test (Fig. 16); cysts were
large, spherical and thick-walled (Fig. 17).
Test dimensions from the 39 specimens of N. ki-
vuense measured in the Ontario samples (Table 1) agree
well with those given by Gauthier-Lièvre and Thomas
(1961); their values being, lengths = 58–63 µm, widths
Fig. 1. A reproduction of Figs 1e and 1f from Gauthier-Lièvre and
Thomas (1961) illustrating the shape of the test of Nebela kivuense;
a and c – lateral (edge) views; b and d – plan views.
= 18–22 µm, and pseudostomal apertures = 13–15 µm.
It might be argued that the Ontario specimens were
larger than those in the type material, but this difference
is not likely signicant if the number of specimens from
the type material was much lower (these data were not
given by Gauthier-Lièvre and Thomas).
The other difference between the two populations
relates to the relative curvature of the tests. Most of the
tests in the African samples were apparently slightly
curved, while only a few of the Ontario specimens
were curved. These differences are also considered to
be within the range of normal intra-specic variation,
owing to the great similarity in general size and shape
of the tests of the two populations. Most notable also,
is the agreement in the apparent selection of both large
and small scales of “foreign” testate amoebae used to
construct the N. kivuense tests in both the African and
Canadian populations. N. kivuense appears to have of-
ten preferred the larger elliptical or circular scales of
Puytoracia, Sphenoderia and large Trinema species
(Figs 18–21). This is in marked contrast to the scales
appearing in the tests of many other much larger Nebela
species which tend to select the smaller scales of other
testate amoebae species (see, for example, the SEM im-
ages of several Nebela species in Kosakyan et al. 2012).
In both the African and Canadian populations, there
was a copious application of organic cement securing
the testacean scales to the integument of the N. kivuense
tests. This was applied often along straight lines with-
out following the curved edge of the scales such that the
shape of the scales observed with lower magnication

Nebela kivuense in the “New World” 285
Table 1. Descriptive statistics (measures of central tendency) for test lengths, widths and test aperture width in Nebela kivuense and Nebela
militaris (Ap. Width = width of the pseudostomal aperture; CO-VAR = coefcient of variation). All measurements in µm.
N. kivuense (n = 39) N. militaris (n = 54)
Test Length Test Width Ap. Width Test Length Test Width Ap. Width
55.1 23.0 13.8 MEAN 72.0 39.7 19.6
55 23 14 MEDIAN 72 40 20
49 20 12 MINIMUM 64 34 17
64 26 16 MAXIMUM 82 46 22
6.1 6.2 7.2 % CO-VAR 5.7 7.3 7.1
Figs 2–17. Diagramatic representations and light and electron mi-
croscope images illustrating test morphology of Nebela kivuense.
Figs 2–6, 12a, 13a and 14 – variations in test shape as seen in plan
views together with the lateral view of the same tests (Figs 7–11,
12b, 13b). Fig. 15 – scanning electron microscope image showing
the attened anterior region of a test with its thickened rim of or-
ganic cement around the pseudostomal aperture (arrow). Fig. 16 –
well developed internal cyst plate (arrow). Fig. 17 – thick-walled
spherical cyst (arrow).
light microscopy was often perceived to be polygonal
rather than truly circular or elliptical. Gauthier-Lièvre
and Thomas (1961) erroneously described the scales
included in the tests of the African specimens as being
“polygonales irrégulières”, and this feature was repro-
duced in their drawings (Fig. 1). What appeared to be
very thin polygonal plates in some of the Canadian ma-
terial, were revealed to be circular or elliptical scales,
after dissolution of the organic cement in concentrated
HNO3 (or after mounting in the high refractive index
media, Styrax or Canada Balsam; Figs 18–21); in no
cases were any polygonal non-scale agglutinated mate-
rials found in tests of this species.
Because errors in identication can lead to false
conclusions about geographic range of testate amoe-
bae and other small protists (Mitchell and Meisterfeld
2005), it is important to ask the question: “is it possi-
ble that N. kivuense is more widespread globally than
the title of this paper suggests, but is ‘hiding’ behind
the name of another similar species?”. It is necessary,
therefore, to draw attention to the features that distin-
guish N. kivuense from other Nebela species. N. ki-
vuense is among the smallest of the known Nebela spe-
cies, so there are only a limited number of options for
its misidentication. Because there was overlap in the
lengths of tests of N. kivuense and N. militaris reported
in the Nebela literature, and because N. miliaris and
N. kivuense co-occurred in some of the Canadian sam-
ples, I compared the tests dimensions in these two
taxa (Table 1; Fig. 22). Despite the minor overlap in
test lengths, these are clearly two separate species on
the basis of pseudostomal aperture and test widths that
were much greater in N. militaris. Furthermore, it can-
not be argued that N. kivuense might be an “ecologi-
cal variant” of N. militaris because populations of both
taxa co-occurred in the same ecosystems.

K. H. Nicholls
286
being confused with other Nebela species in the pub-
lished literature.
The discovery of Nebela kivuense in temperate
North America has implications for biogeography. An
Figs 18–21. Dried tests with circular or elliptical scales “scav-
enged” from other species of testate amoebae. Figs 18, 20 – light
microscope images of tests mounted in Canada Balsam. Figs 19,
21 – scanning electron microscope images of two different tests.
A similar but less rigorous comparison was made of
N. kivuense with ve other Nebela taxa based on lit-
erature descriptions (Table 2). It is apparent that N. ki-
vuense is a unique species that has a low probability of
Table 2. Comparisons of test size and shape in Nebela kivuense with other small Nebela species.
Taxon Test Length (L)
and Width (W) (μm)
Notes
Small unnamed form1 of Nebela minor
Penard, 1883 [described by Penard (1902)]
L = 55–70
W = ?
The degree of taper of the neck region of the test is far greater than that found
in N. kivuense (i.e., the ratio of test width to pseudostome width is much greater
in the Leidy/Penard taxon).
N. gimlii Singer et Lara, 2015: Singer et al.
(2015)
L = 68–78
W = 50–62
Its much wider test and its lack of the elongated tapered neck region of the test
set this apart from of N. kivuense.
Nebela cylindrica Bonnet, 1979: Bonnet
(1979)
L = 48–50
W = 17–22
It cannot be confused with N. kivuense because its test is only slightly attened
(hence the specic epithet, “cylindrica”) with a thin poorly developed collar
surrounding the pseudostomal aperture.
Nebela tubulata Brown, 1911; (assigned
to Padaungiella, Lara et Todorov [in Ko-
sakyan et al. 2012]); (Mitchell, 2003;
http://istar.wikidot.com/id-keys/Nebela)
L = 55–74
W = 28–48
This species has a prominent anterior portion with nearly parallel sides and
a markedly bulbous and much wider posterior; there is no possibility of taxo-
nomic overlap with N. kivuense.
Nebela militaris Penard, 1890; see also
Penard (1902) and Deandre (1936)
L = 50–72
W = 25–38
The test shape is noticeably more “bulbous” in plan view in N. militaris (i.e.,
has a much lower length-to-width ratio than in N. kivuense; see also Fig. 22).
Nebela militaris var. curvata Golemansky,
1962
L = 90
W = 40
This taxon was described (Golemansky, 1962) as a large N. militaris with
a markedly curved test. Its much larger size readily distinguishes it from N.
kivuense.2
1 Penard (1902) said that this taxon was likely the same as the form illustrated in Leidy’s (1876) Plate XXII, Figs 11, 12 and 16, which Leidy had inappro-
priately lumped in with N. collaris Ehrenberg. The modern concept of N. collaris, however, includes only forms with test lengths in the range of 95–115 µm
(Kosakyan et al. 2013). This leaves out the small “form” described by both Leidy and Penard.
2 Also, there have been no previous reports of N. militaris var. militaris with slightly curved tests in plan view or tests with false-polygonal plate structures
(as described above for N. kivuense) in either N. militaris var. militaris or N. militaris var. curvata.
Fig. 22. Comparison of test length, width and pseudostomal aper-
ture width in Nebela kivuense and N. militaris, co-occurring in the
Ontario (Canada) samples.

Nebela kivuense in the “New World” 287
argument has been made that there are few barriers to
the global distribution of small, single-celled protists
(Fenchel and Findlay 2004). Owing to the relative ease
by which small organisms and their cysts are blown by
wind or transported long distances in the plumage of
water birds, there would appear to be a size threshold
above which these transport vectors are much less effec-
tive. A prevailing hypothesis suggesting that small pro-
tists are more likely to be “everywhere”, while protists
generally above a size of 100–150 µm tend to exhibit
more regional tendencies in their world distributions
(Wilkinson 2001, Yang et al. 2010). It has also been ar-
gued that there are truly rare species whose distribution
is controlled by special environmental tolerances and
requirements, irrespective of cell size (Foissner 2007);
such species will include endemic species that are par-
ticularly adapted to polar, high alpine, or other “harsh”
or unique environments.
The type locality for Nebela kivuense as described
by Gauthier-Lièvre and Thomas (1961) was an area of
wet moss between Lubèro and Butembo in the Demo-
cratic Republic of the Congo (DRC). This is about 50
km E of Lake Edward, about 150 km N of Lake Kivu
on the border between the DRC and Rwanda. Lake
Edward lies on the Equator at an elevation of 920 m,
and experiences typical African equatorial climate with
a short rainy season in October and a longer rainy sea-
son in April and May. Although the original collection
site was not described in any detail, the type locality
for N. kivuense was most probably along the main road
north of Lubèro, that skirts the western boundary of
Virunga National Park and passes through the south-
western foothills of the Rwenzori Mountains (“Moun-
tains of the Moon”). The equatorial climate at the el-
evation of Lake Edward would be moderated towards
a slightly more sub-tropical climate on the lower slopes
of these mountains.
In marked contrast, the climate of the area near the
Canadian habitats of N. kivuense, at 44° North Latitude,
was characterized by a winter period from Decem-
ber to March with an average temperature of –4.7°C
(1981–2010) when the ground is frozen and covered
with snow. Total annual average precipitation for this
region of Ontario (Udora weather station) was 885 mm
(82% rain, 18% snow, as water equivalent). The daily
average temperature for the warmest month (July) was
20°C and –7°C for the coldest month (January).
Until now, it could be assumed from the limited
data on N. kivuense, that it might be characterized as
a very rare tropical species. More likely, based on the
recent discovery in Canada, it would appear to have
a wide climate tolerance and could be expected to
grow in many moist, moss-dominated habitats in both
temperate and equatorial locations. The lack of reports
of this species do, however, suggest that it is a very
rare species; and the Canadian discovery further sug-
gests that very rare species masquerading as endemics
can achieve global distributions and remain regionally
rare.
Acknowledgements. I thank Ranganathan Santhanam, Univer-
sity of Ontario, Institute of Technology for assistance with electron
microscopy.
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Received on 11th February, 2015; revised on 27th February, 2015;
accepted on 13rd March, 2015