First records of Protosteloid Amoebae (Eumycetozoa) from the Democratic Republic of the Congo

Abstract

Background: The first records of Protosteloid Amoebae in the Democratic Republic of the Congo are discussed in the present paper. This survey on Protosteloid Amoebae is the first from Central African; the previous records for the African continent were restricted to Egypt, Kenya, Malawi, Tanzania and Uganda.
Methods: Aerial litter samples, collected in 2010 during the “Boyekoli Ebale Congo” expedition in the Congo River basin between the cities of Kisangani and Bumba, were put into culture on wMY medium, a weak malt yeast nutrient agar medium.
Results: The aerial litter cultures revealed 23 species representing 70 % of the total number of species described worldwide. Two of these taxa, Schizoplasmodiopsis reticulata and Schizoplasmodium seychellarum, are new records for the African continent. The isolate LHI05 was observed for the first time on a substrate collected outside Hawai’i. In addition, 5 unknown taxa were observed. A selection of micrographs is presented of the new recorded species, the unknown taxa and all their related species observed in this study.
Conclusion: The high species diversity observed on a limited number of samples suggests that the investigated region is, together with Hawai’i, one of the world’s tropical hotspots for Protosteloid Amoebae.

Full text

Plant Ecology and Evolution 147 (1): 85–92, 2014
http://dx.doi.org/10.5091/plecevo.2014.883
First records of Protosteloid Amoebae (Eumycetozoa)
from the Democratic Republic of the Congo
Myriam de Haan1,*, Christine Cocquyt1, Alex Tice2, Geoff Zahn2 & Frederick W. Spiegel2
1Botanic Garden Meise, Nieuwelaan 38, BE-1860 Meise, Belgium
2Department of Biological Sciences, SCEN 601, 1 University of Arkansas, Fayetteville, Arkansas 72701, USA
*Author for correspondence: myriam.dehaan@br.fgov.be
INTRODUCTION
Protosteloid Amoebae, formerly known as protostelids (Ol-
ive 1967), represent a small group of thirty three species de-
scribed worldwide (Spiegel et al. 2007b). Identication of the
taxa is primarily based on morphologic characters of the mi-
croscopic fruiting bodies or sporocarps, composed of a trans-
lucent stalk supporting one or more spores. Protostelids were
traditionally placed together with and considered as progeni-
tors of two other groups of fruiting amoebae, myxomycetes
and dictyostelids in the Eumycetozoa (Olive 1975). Recent
molecular phylogenetic research by Shadwick et al. (2009)
revealed that protostelids are not monophyletic and that only
a few of them are related to myxomycetes and dictyostelids.
Furthermore the study showed that the protostelids can be
divided into at least seven non-related but well supported
clades, some showing afliations with known groups of non-
fruiting amoebae, scattered throughout the Amoebozoa. For
this reason it was proposed to replace the name protostelids
by Protosteloid Amoebae (Shadwick et al. 2009).
Data about the distribution and ecology of these protists
is not spread evenly throughout the world. North America
and Europe are relatively well studied, but from other tem-
perate regions less data are available (Ndiritu et al. 2009, de
Haan 2011). Most parts of the tropics are still unexplored.
A few surveys have been published from South and Cen-
tral America, e.g. Costa Rica (Stephenson & Moore 1998,
Moore & Stephenson 2003) and Puerto Rico (Stephenson et
al. 1999, Moore & Spiegel 2000). Data have been published
from Australia (Powers & Stephenson 2006), and results
from a series of surveys from Hawai’i have been presented
(Spiegel et al. 2007a). Few records were known from Africa,
restricted to Egypt and Uganda (Olive & Stoianovitch 1969,
1972, 1976) until recently Ndiritu et al. (2009) published a
large survey carried out in the Aberdare region, Kenya, in-
cluding some data from Malawi and Tanzania.
The present paper deals with the rst records of Protoste-
loid Amoebae from the Democratic Republic of the Congo
(D.R. Congo).
Aerial litter samples were collected during a multidisci-
plinary expedition in D.R. Congo in May and June 2010. The
expedition was an initiative of the Congo 2010 Consortium
composed of three Belgian and two Congolese institutes, the
All rights reserved. © 2014 Botanic Garden Meise and Royal Botanical Society of Belgium – ISSN 2032-3921
REGULAR PAPER
Background – The rst records of Protosteloid Amoebae in the Democratic Republic of the Congo are
discussed in the present paper. This survey on Protosteloid Amoebae is the rst from Central Africa; the
previous records for the African continent were restricted to Egypt, Kenya, Malawi, Tanzania and Uganda.
Methods – Aerial litter samples, collected in 2010 during the “Boyekoli Ebale Congo” expedition in the
Congo River basin between the cities of Kisangani and Bumba, were put into culture on wMY medium, a
weak malt yeast nutrient agar medium.
Results – The aerial litter cultures revealed 23 species representing 70% of the total number of species
described worldwide. Two of these taxa, Schizoplasmodiopsis reticulata and Schizoplasmodium
seychellarum, are new records for the African continent. The isolate LHI05 was observed for the rst
time on a substrate collected outside Hawai’i. In addition, 5 unknown taxa were observed. A selection
of micrographs is presented of the new recorded species, the unknown taxa and all their related species
observed in this study.
Conclusion – The high species diversity observed on a limited number of samples suggests that the
investigated region is, together with Hawai’i, one of the world’s tropical hotspots for Protosteloid Amoebae.
Key words – Aerial litter, Central Africa, D.R. Congo, Eumycetozoa, Protosteloid Amoebae, Species
richness.

86
Pl. Ecol. Evol. 147 (1), 2014
Royal Museum for Central Africa, the Royal Belgian Insti-
tute of Natural Sciences and the National Botanic Garden
of Belgium, the University of Kisangani and the National
Herbarium of D.R. Congo at Yangambi (www. congobiodiv.
org). It was organized in 2010 on the occasion of the interna-
tional year of biodiversity and of the 50th anniversary of the
independence of D.R. Congo. The expedition was given the
title “Boyekoli Ebale Congo”, Lingala for “the study of the
Congo River” (Congo Biodiversity Initiative 2010). The aim
of the expedition was to assess the biodiversity in and along
the Congo River between the cities of Kisangani and Bumba
in locations with almost no to extreme anthropogenic impact.
The aim of the present study was to ascertain whether Pro-
tosteloid Amoebae were present in the studied locations and
if so, to obtain the highest possible number of species from
the chosen substrate type – aerial litter. An important goal
was to observe rare taxa, because little is known of their mor-
phological variation, distribution and substrate preferences.
MATERIALS AND METHODS
The region visited during the expedition in 2010 is located
in the Congo River basin between the cities of Kisangani
(Tshopo district, Orientale province) and Bumba (Mongala
district, Équateur province) (Virgilio et al. 2011). Additional
localities along three of the major tributaries of the Congo
River, the rivers Itimbiri, Lomami and Aruwimi, were also
explored. The sampling locatities were situated at an eleva-
tion of 357–382 m above sea level. The climate in this region
is tropical, hot and humid, type Af of the Köppen-Geiger
climate classication system (Kottek et al. 2006), the aver-
age annual maximum temperature is 29°C, the minimum
21°C, and the annual relative humidity is 86%. The average
monthly precipitation in Basoko, a town situated in the cen-
ter of the visited region, is highest in April (200 mm) and in
October (216 mm), the driest month is January with an aver-
age of 70 mm (data from www.weatherbase.com). Sampling
took place in May (avg. ppt. 176 mm), towards the end of the
rainy season.
Samples were taken at ve localities: Yaekela, Engengele,
Kona 1, Kona 2 and Yara. The habitats ranged from tropical
lowland rainforest with no, almost pristine, over moderate to
extensive anthropogenic impact (table 1). We considered oc-
casional selective tree cutting as moderate, and forest fallows
as extensive anthropogenic impact.
Thirteen samples of aerial litter or dead primary tissue
still attached to living plants for the culturing of Protosteloid
Amoebae were collected by M. de Haan and C. Cocquyt
(table 1). Aerial litter and ground litter are the microhabitats
with the highest species abundance and diversity of Proto-
steloid Amoebae (Moore & Spiegel 2000, Spiegel et al.
2004). As such for this survey aerial litter was given prefer-
ence to ground litter. Moreover cultures of the latter substrate
type are known to be very rapidly overgrown with moulds
thus hindering observation. All collected material was air-
dried on site in separate paper bags. Each sample was put
into culture and examined in the laboratory of the National
Botanic Garden of Belgium by M. de Haan. Duplicates of
the samples were sent to the Department of Biological Sci-
ences, University of Arkansas (USA) to be cultured and ex-
amined by A. Tice, G. Zahn and F.W. Spiegel.
The method of culturing by Spiegel et al. (2007b) after
Olive (1975) was followed. Each substrate sample was cut
into pieces of about 1 cm wide and 2 cm long and soaked
in sterile water for 30 min. About eight pieces or strips per
sample were distributed evenly on a 9 cm Petri dish contain-
ing a weak malt yeast nutrient agar medium (1 liter of wMY
medium: 0.002 g malt extract, 0.002 g yeast extract, 0.75 g
K2HPO4 and 15 g agar in distilled water) representing one
primary isolation plate (PIP). For each sample a minimum of
three PIPs were prepared for observation in order to obtain
as many taxa as possible.
After an incubation-period of three days at room temper-
ature the PIPs were examined under a compound microscope
(x200 and x400 magnication) every three days during the
rst three weeks, once a week for the next 8 weeks and from
then on once a month. Photographs were taken by M. de
Haan with a digital single-lens reex camera (Olympus E-3)
mounted on the microscope (Pro.Way, BK 5000) using plan
innity x20 or x40 objectives. A copy of all images taken
during this study will be deposited at the National Botanic
Garden of Belgium and the “Centre de Surveillance de la
Biodiversité” (CSB) in Kisangani (D.R. Congo). The collect-
ed samples are deposited in the National Botanic Garden of
Belgium and duplicates of these samples in the Department
of Biological Sciences, University of Arkansas, USA.
Results of the PIP counts performed at the laboratory of
the National Botanic Garden of Belgium and at the depart-
ment of Biological Sciences, University of Arkansas (USA)
were collated. Species abundances were determined and
divided in four classes according to Ndiritu et al. (2009). A
record of a taxon on one strip corresponds with a positive ob-
servation made in one eld of view. The relative abundance
is the percentage of the number of records made on all strips
and is divided by total number of strips made on the PIPs
from all the samples. Relative abundance classes are > 10%
abundant (A); 10-5% common (C); 4.9–1% occasional (O);
< 1% rare (R).
The names of the Protosteloid Amoebae and myxomy-
cetes species follow the nomenclature proposed by Lado
(2005–2013). The names of the substrate species are consis-
tent with the nomenclature of The Plant List (2010–2013).
RESULTS AND DISCUSSION
The taxa observed during the present study are listed in table
2 with their respective relative species abundance and occur-
rence on the specic substrate samples. The different sub-
strate species are presented per locality in table 1, with the
respective Protosteloid Amoebae taxa observed on the PIPs.
A selection of micrographs of the new recorded species, the
unknown taxa and all their related species observed during
this study, is provided on gure 1.
A total of twenty two species of Protosteloid Amoebae
and one minute myxomycete, Echinostelium bisporum,
which is usually treated as a Protosteloid Amoeba (Ndiritu
et al. 2009), were observed on the PIPs. This represents 70%
of the thirty three species known worldwide, which is a high

87
de Haan et al., Protosteloid Amoebae from D.R. Congo
Locality Collection
date Coordinates Vegetation Sample
number Substrate Taxa Spp Pm
s.l. N.N.
Yaekela 4 May
2010
0.81183° N
24.28276° E
clearing in tropical lowland
rain forest that is
regularly ooded – 3
MdH-DRC-
Proto001
Placodiscus sp.
(Sapindaceae) Ca, Eb, Eo, Pm, Pn, Sps, Sr, Si 8 1 0
Yaekela 4 May
2010
0.81183° N
24.28276° E
clearing in tropical lowland
rain forest that is
regularly ooded – 3
MdH-DRC-
Proto002
Musanga cecropioides
R.Br. (Utricaceae)
Ca, Eb, Eo, Ez, Mp, Pa, Pm, Pn, Sa,
Se, Sps, Sv, Si, Sp2 13 1 1
Yaekela 4 May
2010
0.81199° N
24.28249° E
small banana plantation in
tropical lowland rain forest – 3
MdH-DRC-
Proto003 Musa sp. (Musaceae)
Ca, Cr, Eb, Eo, Ez, Ng/Ct, Pa,
Partic, Pm, Pmvc, Pml, Pmr, Pn,
Ppyr, Sa, Sps, Sc, Si, Sv, Ta, Sp1,
Sp5
17 4 2
Engengele 10 May
2010
2.05898° N
22.70203° E
ooded river bank with reed
(Phragmites sp.) – 3
MdH-DRC-
Proto004
Irvingia smithii Hook.f.
(Irvingiaceae) non observed 0 0 0
Kona 1 11 May
2010
2.04096° N
22.78805° E tropical lowland rain forest – 2 MdH-DRC-
Proto005
Microsorum
punctatum (L.) Copel.
(Polypodiaceae)
Ca, Cr, Eb, Eo, Ng/Ct, Pa, Partic,
Pm, Pmvc, Pmr, Pn, Sa, Sps, Sr, Sv,
Sc, Ss, Se, Si, Ta, Sp2, Sp3, Sp6
18 3 3
Kona 1 11 May
2010
2.04096° N
22.78805° E tropical lowland rain forest – 2 MdH-DRC-
Proto006
Chromolaena odorata
(L.) R.M.King & H.Rob.
(Compositae)
Ca, Eb, Pa, Pm, Sa, Sps, Se, Si 8 1 0
Kona 1 11 May
2010
2.04096° N
22.78805° E tropical lowland rain forest – 2 MdH-DRC-
Proto007
Rhabdophyllum sp.
(Ochnaceae)
Ca, Eo, Mp, No, Pa, Pm, Sa, Sps, Sr,
Sv, Sc, Se, Si, Ta, Sp4, Sp5 14 1 2
Kona 1 11 May
2010
2.04046° N
22.78787° E tropical lowland rain forest – 2 MdH-DRC-
Proto008
Elaeis guineensis Jacq.
(Arecaceae) Ca, Eo, Pm, Pn, Sps, Si 6 1 0
Kona 1 12 May
2010
2.04096° N
22.78805° E tropical lowland rain forest – 2 MdH-DRC-
Proto011 Musa sp. (Musaceae) Ca, Eb, No, Pa, Pm, Pml, Pn, Sps,
Sc, Si 9 2 0
Kona 2 12 May
2010
2.05188° N
22.79409° E tropical lowland rain forest – 1 MdH-DRC-
Proto009 Cola sp. (Malvaceae) Ca, No, Pm, Sa, Sps, Sv, Sc, Si, Ta 9 1 0
Kona 2 12 May
2010
2.05188° N
22.79409° E tropical lowland rain forest – 1 MdH-DRC-
Proto010 Maranthaceae Ca, Ez, Pa, Pf, Pm, Pml, Sa, Sps, Sv,
Sc, Si, Ta 11 2 0
Kona 2 12 May
2010
2.05604° N
22.79514° E tropical lowland rain forest – 1 MdH-DRC-
Proto012
Millettia sp.
(Leguminosae) Ca, Eb, Mp, Ng/Ct, No, Pm, Ss, Ta 8 1 0
Yara 31 May
2010
0.70273° N
24.20061° E
tropical lowland rain forest,
dominated by
Gilbertiodendron sp. – 1
MdH-DRC-
Proto013 unknown tree Ca, Sa, Sps, Sr 4 0 0
Table 1 – Sample localities with the collection date, coordinates, vegetation type, sample number and its corresponding substrate and the Protosteloid Amoebae recorded
(abbreviations of the taxa are given in table 2), number of species (Spp), taxa of the Protostelium mycophaga complex (Pm s.l.) and unknown taxa (N.N.) per sample.
Level of anthropogenic disturbance indicated as follows in the column with information on the vegetation: 1 = none, 2 = moderate, 3 = extreme.

88
Pl. Ecol. Evol. 147 (1), 2014
Taxa observed in the present study Abbreviation Relative
abundance class
Occurrence on substrate
(MdH-DRC-Proto)
Cavostelium apophysatum L.S.Olive Ca A 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13
Clastostelium recurvatum L.S.Olive Cr O 3, 5
Echinosteliopsis oligospora Reinhardt & L.S.Olive Eo C 1, 2, 3, 5, 7, 8
Echinostelium bisporum (L.S.Olive & Stoian.) K.D.Whitney
& L.S.Olive Eb C 1, 2, 3, 5, 6, 11, 12
Endostelium zonatum (L.S.Olive & Stoian.) W.E.Benn. &
L.S.Olive Ez O 2, 3, 10
Microglomus paxillus L.S.Olive & Stoian. Mp O 2, 7, 12
Nematostelium gracile (L.S.Olive & Stoian.) L.S.Olive or
Ceratiomyxella tahitiensis L.S.Olive & Stoian. Ng/Ct O 3, 5, 12
Nematostelium ovatum (L.S.Olive & Stoian.) L.S.Olive &
Stoian. No O 7, 9, 11, 12
Protosporangium articulatum L.S.Olive & Stoian. Partic R 3, 5
Protosteliopsis micola (L.S.Olive) L.S.Olive & Stoian. Pf R 10
Protostelium arachisporum L.S.Olive Pa A 2, 3, 5, 6, 7, 10, 11
Protostelium mycophagum var. mycophagum L.S.Olive &
Stoian. Pm A 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12
Protostelium mycophagum var. crassipes L.S.Olive & Stoian. Pmvc R 3, 5
Protostelium mycophagum type little Pml O 3, 10, 11
Protostelium mycophagum type repeater Pmr O 3, 5
Protostelium nocturnum Spiegel Pn O 1, 2, 3, 5, 8, 11
Protostelium pyriformis L.S.Olive & Stoian. Ppyr R 3
Schizoplasmodiopsis amoeboidea L.S.Olive & K.D.Whitney Sa C 2, 3, 5, 6, 7, 9, 10, 13
Schizoplasmodiopsis pseudoendospora L.S.Olive, M.Martin.
& Stoian. Sps A 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13
Schizoplasmodiopsis reticulata L.S.Olive & Stoian. Sr O 1, 5, 7, 13
Schizoplasmodiopsis vulgaris L.S.Olive & Stoian. Sv O 2, 3, 5, 7, 9, 10
Schizoplasmodium cavostelioides L.S.Olive & Stoian. Sc O 3, 5, 7, 9, 10, 11
Schizoplasmodium seychellarum L.S.Olive & Stoian. Ss O 5, 12
Soliformovum expulsum (L.S.Olive & Stoian.) Spiegel Se O 2, 5, 6, 7
Soliformovum irregularis (L.S.Olive & Stoian.) Spiegel Si A 1, 2, 3, 5, 6, 7, 8, 9, 10, 11
Tychosporium acutostipes Spiegel, D.L.Moore & J.Feldman Ta C 3, 5, 7, 9, 10, 12
Proto-DRC-Sp1 Sp 1 R 3
Proto-DRC-Sp2 = LHI05 Sp 2 O 2, 5
Proto-DRC-Sp3 Sp 3 R 5
Proto-DRC-Sp4 Sp 4 R 7
Proto-DRC-Sp5 Sp 5 O 3, 7
Proto-DRC-Sp6 Sp 6 R 5
Table 2 – List of the taxa observed in the present study, conventional abbreviations of each of the recorded taxa, relative abundances
divided into four classes: > 10% abundant (A); 10–5% common (C); 4.9–1% occasional (O); < 1% rare (R) (abundance classes
according to Ndiritu et al. 2009) and occurrence on the specic substrate samples.
The numbers of the substrates correspond with the substrate number as given in table 1.
yield considering that only thirteen samples were collected. A
few isolated records are known from Egypt and Uganda (Ol-
ive & Stoianovitch 1969, 1972, 1976). The only other pub-
lished study on Protosteloid Amoebae from Africa (Ndiritu
et al. 2009) is a large survey in East Africa, where eighteen
species (including E. bisporum) were observed on forty six
aerial litter samples, and a total of twenty one when includ-
ing the results from forty nine ground litter and forty nine
bark samples. Other surveys in tropical forests of Costa Rica
(Stephenson & Moore 1998, Moore & Stephenson 2003),
Puerto Rico (Stephenson et al. 1999, Moore & Spiegel 2000)
and Australia (Powers & Stephenson 2006) have produced
sixteen, thirteen and eight species respectively. Presently
Hawai’i holds the highest species diversity; all thirty three
described Protosteloid Amoebae have been recorded from
this tropical island (Spiegel et al. 2007a).
An average of 9.5 species (range 0–18) was observed on
the thirteen substrates from D.R. Congo which is very high.
When the unknown taxa are included the average increases
to 10.1 (range 0–21). Three plates were prepared per sample.

89
de Haan et al., Protosteloid Amoebae from D.R. Congo
Figure 1 – Images taken from the PIPs of the new records for Africa, of the unknown taxa and their related species observed in this
study: A–E, Schizoplasmodium seychellarum; F, Schizoplasmodiopsis reticulata; G & H, Proto-DRC-sp1; I, Microglomus paxillus; J & K,
Cavostelium apophysatum; L–N, Proto-DRC-sp2; O, Proto-DRC-sp6; P, Protosteliopsis micola; Q–S, Clastostelium recurvatum; T, Proto-
DRC-sp3; U & V, Proto-DRC-sp4; W & X, Proto-DRC-sp5. Scale bar = 10 µm, except D = 50 µm.

90
Pl. Ecol. Evol. 147 (1), 2014
When the substrates yielded an initial high species number
and/or interesting taxa, additional plates were set up to con-
rm observations. In a few cases more taxa were added to
the species list of a particular substrate. A reason for the high
overall species yield could be the ideal weather conditions at
the time of sampling, with heavy rainfall every 3 to 5 days
and dry weather in between. Most samples were collected
2 to 3 days after rainfall. The only sample from which no
observations of Protosteloid Amoebae or myxomycetes were
made originated from a tree, Irvingia smithii, growing on the
ooded bank of the river Itimbiri. Apparently the collected
leaves were still too fresh, there was still a green hue from
chlorophyll that was not entirely degraded. All cultures made
from this sample were overgrown with moulds within a few
days, probably due to the constant high humidity of the sub-
strate.
Substrates from different plant species supported vary-
ing number of Protosteloid Amoebae. Highest species num-
bers were obtained from Microsorium punctatum, Musa sp.
(banana tree), and Rhabdophyllum sp. respectively yielding
eighteen, seventeen and fourteen species, and in addition
three, two and two of the unknown taxa respectively (ta-
ble 1). Musa sp. was collected in two different locations: one
from a small eld in the forest near the village of Yaekela
with extensive anthropogenic inuence and another one from
Kona 1 which is situated about 215 km north-west from
Yaekela, at the entrance of the lowland tropical forest with
moderate human interference, i.e. occasional selective tree
cutting. This sample from Kona 1 yielded nine species. At
the moment it is not possible to attribute the role of human
interference in these locations when it comes to determining
species diversity of Protosteloid Amoebae (see Ndiritu et al.
2009).
Below an annotated account is given of each species en-
countered during the present study, with special attention to
their taxonomy, distribution and ecology.
Cavostelium apophysatum is the most common species
recorded in the study, present on twelve of the thirteen sub-
strates, closely followed by Protostelium mycophagum sensu
lato and Schizoplasmodiopsis pseudoendospora each present
on eleven samples. Soliformovum irregularis was observed
on ten substrates (table 1). C. apophysatum is one of the
most common species in tropical areas (Ndiritu et al. 2009),
which is conrmed in the present study. Schizoplasmodiop-
sis pseudoendospora and Soliformovum irregularis are both
very common in surveys from all parts of the world. The for-
mer species tends to prefer higher temperatures and the latter
shows a preference for a cooler and wetter climate (Ndiritu et
al. 2009, Aguilar et al. 2011).
Protostelium mycophagum is common in all climate
zones especially on aerial litter, but most likely it represents
a complex comprising subspecic taxa and/or cryptic species
(Spiegel et al. 2007b, Ndiritu et al. 2009). Further in-depth
molecular phylogenetic study will reveal more on the pres-
ence or absence of possible cryptic species. One variety, P.
mycophagum var. crassipes and at least two distinct forms,
conventionally designated as Pm “little” and Pm “repeater”
(Spiegel et al. 2007b) were observed respectively three and
two times on different substrates (table 2). P. mycophagum
var. mycophagum is usually observed forming large colonies,
although in this study only small colonies of a few dozen and
even solitary fruiting bodies were seen.
Protostelium arachisporum is more common in tropical
areas (Ndiritu et al. 2009), and was observed on seven sub-
strates. Different forms of P. arachisporum with differences
in spore shape and stalk length were observed in isolated
colonies on four of the substrates. Here too, phylogenetic re-
search is needed to demonstrate whether it concerns a very
variable species or a species complex.
Protostelium pyriformis is not uncommon in tropical re-
gions. However, it was found in our study on only one sam-
ple, a banana tree from Yaekela.
Another species that is expected to be more common in
samples from the tropics is Nematostelium gracile. Aguilar
et al. (2011) noted that this species shows a preference for
high annual but seasonal rainfall. In the present study it was
observed in three samples, Musa sp. from Yaekela, Microso-
rium punctatum and Millettia sp. from Kona 1 and 2 respec-
tively. These results are far from conclusive because we were
not able to differentiate Nematostelium gracile from Ceratio-
myxella tahitiensis without the trophic stage in culture.
Two rare species, Schizoplasmodiopsis reticulata and
Schizoplasmodium seychellarum, were observed for the rst
time on substrates from Africa. Schizoplasmodiopsis reticu-
lata (g. 1F), 20–35 µm tall, was recorded on dead leaves
from three substrates in three locations. The rst substrate,
a tree belonging to the genus Placodiscus, was collected in
the woodland near the village of Yaekela in a clearing with
extensive anthropogenic impact, e.g. deforestation through
tree cutting and shrub burning. The area is regularly ooded
by the Congo River during the rainy seasons. At the time of
sampling the ground was still drying. The other two sub-
strates, the epiphytic fern Microsorium punctatum and a tree
species, Rhabdophyllum sp., were collected in the location
Kona 1. The third substrate is an unknown tree from a forest
dominated by Gilbertiodendron sp. near the village Yara on
the opposite bank of the Congo River from Yaekela. Schizo-
plasmodiopsis reticulata is close to Schizoplasmodiopsis
vulgaris, a much more common species (Spiegel & Feldman
1993). Both species are found on the same substrates. The
spore of S. reticulata (g. 1F), 9–12 µm in diameter, has a
clearly visible ornamentation of raised ridges. S. vulgaris has
a reticulum with low ridges, which can be seen as angular ir-
regularities on the spore outline. In some of the observations
it was difcult to distinguish these two related species.
Schizoplasmodium seychellarum (g. 1A–E) was ob-
served on two substrates, the epiphytic fern M. punctatum
and a tree species belonging to the genus Millettia, from
Kona 1 and 2. The latter location is a lowland tropical forest
with almost no human impact except for hunting purposes.
The morphology of the fruiting bodies, 50–85 µm tall, was
consistent with the original description (Olive & Stoiano-
vitch 1976). The stalk is robust and almost cylindrical to very
slightly tapering towards the distinct ring shaped apex or ap-
ophysis. The single spore, 20–25 µm in diameter, is loosely
connected to the apophysis with a low ring shaped hylum.
When drying the spore wall becomes dented and wrinkled.
The spores are discharged by means of lateral droplets that

91
de Haan et al., Protosteloid Amoebae from D.R. Congo
swell up and burst. Olive & Stoianovitch (1976) remarked
that the spore discharge is not always successful; this was
indeed observed in our cultures. If there is more than one
droplet or if the single droplet is not placed ideally, the spore
either stays in position or gets stuck somewhere along the
stalk. In that case the spores simply dry up without germinat-
ing. Sometimes the droplets did not burst and due to their
extra weight the spore simply fell from the apophysis. In
most surveys this rare species is found not only in low quan-
tities of colonies but also in less than 1% of the samples, and
prefers aerial litter to other substrates (Spiegel et al. 2007b).
In both of our observations the large fruitings occurred in a
group of about twenty fruiting bodies for a period of no more
than two days on one strip of the substrate. It was only ob-
served in PIPs prepared in the laboratory of the National Bo-
tanic Garden of Belgium.
The opposite was the case with Endostelium zonatum and
Protosporangium articulatum both observed exclusively in
the laboratory of the University of Arkansas. E. zonatum was
found on three substrates, Musanga cecropioides, Musa sp.
both from Yaekela and an unidentied species of the Ma-
ranthaceae family collected in Kona 2. Here also the time of
observation was crucial, as E. zonatum develops in a limited
period of time and is very deciduous. P. articulatum is the
only species in this genus that is occasionally found on aerial
litter; the other four species are known to develop only on
bark and wood.
One of the unknown taxa, Proto-DRC-sp1 (g. 1G & H),
9–11 µm tall, observed on leaves from a banana tree collect-
ed near the village of Yaekela, is morphologically similar to
Cavostelium apophysatum (g. 1J & K) but its sporangium,
8–9 µm in diameter, appears to have multiple spores. Proto-
DRC-sp1 could also be related to small fruitings of the mul-
tiple spored Microglomus paxillus (g. 1I) but the stalks are
narrow cylindrical and well developed whereas the sporangia
of Proto-DRC-sp1 seem to be sessile and resting on a broad
base. Until it is observed again in more detail and, prefer-
ably, isolated into culture, it is difcult to determine whether
its similarity to C. apophysatum or M. paxillus is due to close
relationship or just supercial.
The second unknown taxon observed during this study
and annotated as Proto-DRC-sp2 (g. 1L–N), 25–30 µm tall,
was reported for the rst time by Shadwick et al. (2009) as
isolate LHI05, from ground litter of mixed vegetation col-
lected in native dry/mesic forest in Hawai’i. LHI05 is most
likely a new species. Phylogenetic study (Shadwick et al.
2009) revealed a strong afliation to the Acanthamoebidae,
more specic to the non-fruiting Protacanthamoeba bohemi-
ca Dyková, Veverková-Fialová, Fiala & Dvořáková. Consis-
tent characteristics of this deciduous taxon are the pyriform
spore, 11.5–12.5 × 13–14 µm, with a distinct, narrow, cy-
lindrical hylum and a short and tapering stalk, and the apex
seems to inate before spore discharge. After the spore is re-
leased thorn-like, empty stalks remain on the substrate.
Proto-DRC-sp3 (g. 1T), 35–40 µm tall, isolated from
Microsorium punctatum seems to be a form of the uncom-
mon Clastostelium recurvatum with an elongated upper stalk
segment (g. 1Q–S). This taxon was observed on the same
substrate with its typically short, curved, stalked form, 15–20
µm tall. The spore of C. recurvatum is discharged through in-
ation and rupture of the upper stalk segment, but this could
not be observed in either form during the present study. In
both cases the sporangium, 9–12 µm in diameter, showed the
distinct droplet or pear shaped deformation due to the pres-
ence of usually two, exceptionally three to four spores. C.
recurvatum tends to be more common in the tropics than in
temperate regions; it was found on only two of the substrates
from D.R. Congo and was only observed in the laboratory of
the National Botanic Garden of Belgium.
A single sporangium of isolate Proto-DRC-sp4 (g. 1U,
V), 27 µm tall, found on Rhabdophyllum sp., can be related
to C. recurvatum and Proto-DRC-sp3. The long stalk shows
almost no curvature, possibly because the upper section is in-
ated and preparing to discharge the slightly turbinate spore,
9 × 9.5 µm. In this state the isolate strikingly resembled the
equally erect fruiting body as shown in gure 9 accompany-
ing the original description of C. recurvatum (Olive & Stoia-
novitch 1977).
Proto-DRC-sp5 (g. 1W, X), 10–22 µm tall, from the
Rhabdophyllum sp. cultures could possibly be an unde-
scribed species of Protosteliopsis. Similar sporangia were
also observed in cultures from Hawai’i (unpublished data).
Aerial litter samples from Belgium (de Haan 2011) produced
fruiting bodies with similar features and the stalk in particu-
lar has the same gelatinous appearance as that seen in Proto-
steliopsis species. The broadly turbinate spores, 5–8 µm in
diameter, seem to be ornamented with a broad-meshed low
reticulum. The sometimes faintly curved but always rigid
stalk is almost isodiametric from base to apex.
Proto-DRC-sp6, (g. 1O), 40–60 µm tall, was observed
in the cultures from Microsorium punctatum. The apex of
the long stalk does not narrow abruptly to a ne line, as in
Schizoplasmodiopsis micropunctata L.S.Olive & Stoian. or
Tychosporium acutostipes. Proto-DRC-sp6 resembles close-
ly Protosteliopsis micola (g. 1P), 15–25 µm tall, which
was observed once on a Maranthaceae liana in Kona 2. The
stalk of Proto-DRC-sp6 and P. micola is refractile, having a
gelatinous aspect except for a cylindrical broadening in the
lower part of the stalk. In both taxa the single spore, 8–12
µm in diameter, is spherical and does not appear to have an
ornamentations. Normally P. micola is found on dung, but
there are records from aerial litter (Ndiritu et al. 2009). The
fruiting bodies observed in the survey from Kenya (Ndiritu
et al. 2009) and identied as P. micola, had long stalks, sim-
ilar to those of Proto-DRC-sp6.
CONCLUSIONS
All Protosteloid Amoebae records made in this study are
new for the D.R. Congo and for Central Africa. Moreover
two rare species, Schizoplasmodiopsis reticulata and Schizo-
plasmodium seychellarum, are new records for the African
continent. The twenty three species observed on the PIPs
represent 70% of the number of species described world-
wide. Although the thirteen samples collected are too few for
a statistical analysis, some conclusions can be drawn from
the observations of the D.R. Congo data. Our results dem-
onstrate that the number of plates per sample is crucial to
obtain the highest species diversity and number of colonies.

92
Pl. Ecol. Evol. 147 (1), 2014
Also important is the time spent on the observation of the
plates, including the length of time per observation session,
the number of sessions and most of all the timing of the ob-
servation. Some species develop and stay visible for only a
short period.
No obvious difference in species composition could be
observed between substrates collected in habitats with high
anthropogenic impact and those with almost no human in-
terference.
More morphological, ecological and phylogenetic data
are needed to conclude whether the ve unknown taxa can
be described as new or assigned to known species.
The visited region in the D.R. Congo merits further study
and represents, together with Hawai’i, one of the world’s
tropical hotspots for Protosteloid Amoebae.
ACKNOWLEDGEMENTS
The Boyekoli Ebale Congo 2010 expedition, organized by
the Congo 2010 Consortium, was funded by the Belgian
Development Aid, the Belgian Science Policy and the Na-
tional Lottery of Belgium. We greatly appreciate the useful
suggestions and language corrections by Jonathan C. Taylor
and Ann Bogaerts. We are grateful to Olivier Lachenaud for
the identications of the aerial litter substrates and we thank
our local Congolese guides Mopero (Yaekela) and Tonton
(Kona).
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