The ISME Journal (2013), 1–9
& 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13
www.nature.com/ismej
ORIGINAL ARTICLE
Using environmental niche models to test the
‘everything is everywhere’ hypothesis for Badhamia
Marı́a Aguilar1,2, Anna-Maria Fiore-Donno3, Carlos Lado1 and Thomas Cavalier-Smith4
1
Mycology Department, Real Jardı´n Botánico, CSIC, Madrid, Spain; 2Department of Cell Biology,
Medical Sciences, University of Alberta, Edmonton, AB, Canada; 3Zoology Institute, University
of Cologne, Cologne, Germany and 4Zoology Department, University of Oxford, Oxford, UK
It is often discussed whether the biogeography of free-living protists is better explained by the
‘everything is everywhere’(EiE) hypothesis, which postulates that only ecology drives their
distribution, or by the alternative hypothesis of ‘moderate endemicity’ in which geographic barriers
can limit their dispersal. To formally test this, it would be necessary not only to find organisms
restricted to a geographical area but also to check for their presence in any other place with a similar
ecology. We propose the use of environmental niche models to generate and test null EiE
distributions. Here we have analysed the distribution of 18S rDNA variants (ribotypes) of the
myxomycete Badhamia melanospora (belonging to the protozoan phylum Amoebozoa) using 125
specimens from 91 localities. Two geographically structured groups of ribotypes congruent with
slight morphological differences in the spores can be distinguished. One group comprises all
populations from Argentina and Chile, and the other is formed by populations from North America
together with human-introduced populations from other parts of the world. Environmental climatic
niche models constructed separately for the two groups have significant differences, but show
several overlapping areas. However, only specimens from one group were found in an intensively
surveyed area in South America where both niche models overlap. It can be concluded that
everything is not everywhere for B. melanospora. This taxon constitutes a complex formed by at
least two cryptic species that probably diverged allopatrically in North and South America.
The ISME Journal advance online publication, 17 October 2013; doi:10.1038/ismej.2013.183
Subject Category: Microbial population and community ecology
Keywords: biogeography of protists; moderate endemicity; Myxomycetes; phylogeography
Introduction
The biogeographical patterns of free-living unicellular
eukaryotes are still a subject of debate. The underlying
question is to what extent current geographic barriers
and historical events have restrained the dispersion of
protists, and whether this influence can be traced in
the distribution of the organisms that exist today.
Some authors (Finlay 2002; Fenchel and Finlay 2004)
defend that most of them are cosmopolitan, as
described in the ‘everything is everywhere’ (EiE)
hypothesis. In that case, protist species present in a
given location would be a function of only their
habitat requirements and not of restricted dispersion.
However, others (e.g. Smith and Wilkinson 2007;
Vanormelingen et al., 2008) have found evidence
in favour of the ‘moderate endemicity’ (ME)
hypothesis that at least some organisms have
Correspondence: M Aguilar, Department of Cell Biology,
University of Alberta, 5-31 Medical Sciences Building, Edmonton,
AB T6G 2H7, Canada.
E-mail: aguilarg@ualberta.ca
Received 27 June 2013; revised 3 September 2013; accepted 9
September 2013
geographically restricted distributions (Foissner
1999, 2006; Foissner et al., 2008), not reaching all of
their potentially suitable habitats when they
disperse.
The advent of phylogeographic methods based
on molecular data has improved the resolution for
detecting and analysing variability between
populations and searching for recent dispersal
events. These methods have also shed light on
the existence of cryptic species complexes
(Amato et al., 2007; Smirnov 2007; Bass et al.,
2009; Howe et al., 2009; Morard et al., 2009;
Poulı́čková et al., 2010; Douglas et al., 2011) that
share a common morphology but are genetically
distinct.
Clear molecular evidence of geographically
restricted 18 S rDNA sequence types (ribotypes)
has been found in Foraminifera (Darling et al., 2007;
Aurahs et al., 2009), diatoms (Evans et al., 2009;
Sorhannus et al., 2010) and Cercozoa (Bass et al.,
2007). However, finding a geographic structure does
not allow a full rejection of the EiE hypothesis. It is
still possible that considered lineages are globally
dispersed, but they have different ecological
preferences causing them to inhabit distinct areas.
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One major issue is the limited amount of available
information about protists compared with multicellular organisms. In this context, niche models
can be a valuable tool for generating null
hypotheses of the distribution of the organisms
in an EiE scenario from a limited set of data
(Aguilar and Lado 2012). Environmental niche
models are mathematical functions that describe
the probability of the presence of an organism
according to the values of a number of environmental variables. They are calculated using the
information provided by localities where the
organisms were previously found to make an
extrapolation to other unexplored areas. These
models can be subsequently tested for the
presence of barriers to dispersion by checking
whether organisms have reached all of their
potential habitats.
In this report, we provide a striking example of
geographical genetic differentiation in Myxomycetes (Amoebozoa) that has also been analysed
taking into account the information provided by
niche models. Myxomycetes lend themselves
especially well to such studies as their DNA can
be extracted and sequenced from fruiting bodies
already well preserved in herbaria from many
globally widespread locations. B. melanospora
Speg is an organism usually found on decaying
Cactaceae and other succulent plants. Its fruiting
bodies (sporocarps) are easily visible, as they form
groups of whitish-grey little balls, approximately
1 mm in diameter, that contain dark-coloured,
warted, reticulate spores. The morphology of
B. melanospora is highly variable (Lado et al., 2007),
but it was not previously known whether this
morphological variation is an expression of phenotypic plasticity or is phenotypic evidence of
actual genetic divergence. It appears to be mostly
restricted to the arid regions of America, where it
is very frequent. However, it has never been
collected
from
the
Asian
arid
regions
(Novozhilov et al., 2009), and only rarely from
other parts of the world, including intensively
studied regions like Europe, where it is most
frequently found growing on introduced cactae
(www.gbif.org).
This study reports an analysis of small subunit
ribosomal DNA (SSU rDNA) sequence variation in
B. melanospora on its entire known geographical
range. For a better understanding of the geographical pattern found, clade-specific habitat
preferences have been analysed and compared
using niche models. The main character distinguishing clades, that is, the spore morphology, has
also been studied using s.e.m. Our results are at
odds with the EiE hypothesis, and suggest that
B. melanospora constitutes a geographically
structured complex formed by at least two cryptic
species, which are genetically and morphologically differentiated and display different
distributions.
The ISME Journal
Materials and methods
Sampling, DNA extraction and sequencing
Specimens collected by several myxomycetologists
– C Lado, S L Stephenson, M Meyer, L H Cavalcanti
and R McHugh – comprising more than 10 sporophores were selected for DNA extraction, resulting
in a total of 125 herbarium specimens from 91
different localities (Supplementary Table 1). All
collections were represented by material that fruited
in the field under natural conditions.
DNA was extracted as described elsewhere
(Fiore-Donno et al., 2008), and it was amplified
by polymerase chain reaction (PCR) using
the primers SA’ (TGGTTGATCCTGCCAGTAGTGT)
and
SU19R
(TGTCCTCTAATTGTTACTCGA),
Mangotaq mix (Bioline, London, UK) and the
following cycling parameters: 45 s at 94 1C,
33 (25 s at 94 1C, 60 s at 42 1C, 3.5 min at 72 1C)
and 5 min at 72 1C. To obtain nearly complete SSU
rRNA gene sequences, four overlapping sequence
fragments were obtained using the primers SA0 ,
SU19R, and S4, S900R, S11.5, SR15, DA2, RB2
(Fiore-Donno et al., 2008) and the same polymerase
chain reaction conditions. Purified polymerase
chain reaction products (SureClean kit, Bioline)
were sequenced directly by Macrogen Korea
(Geumcheon-gu, Seoul, South Korea). All new
sequences were submitted to GenBank (accession
numbers on Supplementary Table 2).
Phylogenetic analyses
Sequences were automatically aligned with Geneious 5.4 and the obtained alignment was corrected
by hand. All sites were kept for the analyses. The
best available model of molecular evolution was
selected with MrModeltest 2.3 (Posada and Crandall
1998), and GTR þ G þ I was the best fit. Phylogenetic
trees were primarily constructed using Bayesian
inference (BI), with MrBayes 3.1.2 (Huelsenbeck and
Ronquist 2001). Two identical searches with ten
million generations each (chain temperature ¼ 0.2;
sample frequency ¼ 1000) were conducted. In both
runs, probabilities converged on the same stable
value approximately after generation 8 000 000.
A 50% majority-rule consensus tree was calculated,
and posterior probability (PP) was used as an
estimate of robustness. All BI analyses were
carried out on the freely available Bioportal
(www.bioportal.uio.no). Parameter estimates were
graphically analysed to assess stability with Tracer
1.0.1. Maximum likelihood trees were calculated
with the program RAxML (Stamatakis 2006) using
the model GTRGAMMA for nucleotide substitution,
the rapid hill-climbing (–fd) option and the rapid
bootstrap algorithm (–fa) with 100 bootstrap
replicates.
Ribotype networks, representing unique DNA
sequences separated by mutational steps, were
constructed using statistical parsimony with TCS
software.
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Scanning electron microscopy
Scanning electron microscopy (s.e.m) images were
obtained after the critical-point drying of 32 specimens randomly selected and distributed across the
whole phylogeny, using the s.e.m of the Royal
Botanic Garden of Madrid, employing a Jeol T
330 A s.e.m, at 10–15 kV. The morphology of 10
spores per specimen (320 spores in total) was
studied, and their largest diameter was measured
(Supplementary Table 3). Differences in diameter
length between genetic groups were tested with a
Welsh t-test in R 2.12.2. and a representative spore
(close to the average diameter in the specimen, and
with typical morphology) from each sample was
pictured.
Niche models
Niche models were generated with Maxent 3.3.3a
(Phillips and Dudik 2008) using Bioclim variables
from WorldClim (Hijmans et al., 2005) with a 2.5
arc-minutes resolution and with collections that
were precisely geo-referenced (84 localities).
Bioclim variables represent trends in temperature
and precipitation along the year, as well as seasonality
and extreme or limiting climatic factors. One
locality (coordinates 10.0227781, 30.605471) was
excluded from the analyses as it was not covered
by some environmental layers. The models were
evaluated on the basis of receiver operating characteristic (ROC) analysis, which generates the AUC
(area under the curve) score. To check whether
models from the genetic groups were significantly
different, their outputs were compared using
ENMtools (Warren et al., 2010), with D and I indices
as measures of niche overlap. The D and I values
(Warren et al., 2008) are calculated by comparing
two normalized Maxent environmental niche models,
and using the estimated values of habitat
suitability for each grid. If the niche differences are
not caused only by random effects, the obtained D
and I values from the two original niche models
should display significant differences from the
distribution generated by 100 random pseudoreplicates using pooled samples from the two
original data groups.
Other statistical analyses
Mantel tests of orthodromic geographic distances
versus pairwise raw genetic distances were
performed with the package vegan in R 2.12.2, using
1000 permutations and the Pearson product-moment
correlation coefficient. Geographic distances between
sampling points were previously measured using
the package fields, and genetic distances were
calculated with the package ape (Paradis et al.,
2004). Analyses of molecular variance (AMOVA)
were performed with Arlequin 3.5 (Excoffier et al.,
2005) and significance was based on 10 000
permutations.
3
Results
Bayesian inference (BI)
To assess the genetic structure of populations of B.
melanospora, the largest fragment of the small
subunit ribosomal DNA that is free of type I intron
insertion sites and displays sufficient variability
was amplified. A total of 37 distinct ribotypes with
variable length (510–514 bp) were found in the 125
specimens sequenced, and this short and easy-tosequence gene region showed 87 (16.3%) variable
sites, with an average percent identity of 97.2% and
an average 53.3% GC content. Most mutations were
single nucleotide substitutions. Although there is
still very little information about inter- and intraspecific sequence variability in myxomycetes, we
have used a 97% similarity percentage as a first
rough approximation to a threshold to separate
putative cryptic species, under which samples
clustered defining 6 groups.
Rooting the tree was difficult owing to the low
number of myxomycete accessions in GenBank and
because outgroups have longer branches than B.
melanospora. An initial phylogenetic analysis with
all available nearly complete small subunit ribosomal
DNA sequences of the genera Badhamia and
Physarum, and newly obtained sequences from
seven specimens of B. melanospora and from six
other species of Badhamia and Physarum, was
carried out (Supplementary Figure 1). Lineages most
closely related to B. melanospora were selected as
outgroups for subsequent analyses.
The inferred evolutionary interrelationships
among all ribotypes, using the selected outgroups
and a final alignment of 519 bp with 164 (31.6%)
variable sites, are shown in Figure 1; Supplementary
Table 1 gives sample details. One big paraphyletic
group (A) of 54 sequences (14 ribotypes) is formed
basically by specimens from South America (Chile
and Argentina), but also contains sequences from
Morocco and France. A well-supported divergent
clade (group B) of 71 sequences (23 ribotypes)
groups all North American collections, with two
from Brazil, and most specimens from other parts of
the world (Morocco, Canary Islands, Ascension
Island, Europe, Madagascar).
Phylogenetic network estimation using statistical
parsimony (TCS)
TCS analyses (Figure 1b) were highly congruent
with the results yielded by BI. Five highly divergent
ribotypes (R13, R14, R35, R36 and R37) did not join
the network, but the remaining sequences formed
two well-delimited groups of ribotypes coincident
with the above-defined groups A and B.
Group A has 12 different ribotypes, mostly from
South American populations – Argentina and Chile –
but there are also two from Morocco. One of the
Moroccan ribotypes is shared with Argentinean populations; the other is closely related to the most common
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Figure 1 Phylogenetic analyses. (a): Fifty percent majority-rule rooted consensus tree of a 533 bp fragment of the small subunit rDNA
(SSU) of 125 specimens of B. melanospora obtained by Bayesian inference. Triangles represent collapsed groups of samples with the
same ribotype and geographic origin. Colours indicate the origin of the specimens. The scale bar represents evolutionary distance in
changes per site. Bayesian posterior probabilities and RAxML support values are at each node. (b): Ribotype network of a 533 bp fragment
of small subunit rDNA (SSU) of 123 specimens of B. melanospora. Circle size is proportional to the number of specimens within each
ribotype, and dots between ribotypes represent unobserved, inferred ancestral ribotypes. Lines between ribotypes represent mutational
steps between alleles. Colours denote specimen origin.
Argentinean ribotype. It is remarkable that there is no
ribotype shared by both Argentina and Chile.
Group B has 20 ribotypes, from North America
(USA and Mexico), Brazil and nearly all collections
from the Old World – North Africa, Madagascar and
Europe – and Atlantic oceanic islands – Canary
Islands and Ascension Island. Most ribotypes from
the Old World, Atlantic oceanic islands and Brazil
group together and are closely related to North
American populations.
Table 1 Analyses of Molecular Variance (AMOVA)
NA, SA, OW
NA, SA
NA, OW
SA, OW
FST
% AP
% WP
Tot. Var.
0.51
0.65
0.10
0.53
50.69
65.03
10.40
53.12
49.31
34.97
89.60
46.88
8.83
10.66
5.68
9.29
Statistical analyses
Results obtained after comparing different geographical regions.
Abbreviations: NA, North America; SA, South America; OW, Old
World; FST, fixation index; % AP, percentage of variation among
populations; % WP, percentage of variation within populations; Tot.
Var., total variance. Significance tests after 10000 permutations with
Po0.001 in all cases.
Mantel tests showed that genetic distance is correlated with geographical distance for the American
populations (r ¼ 0.5486, Po0.001), but it is only
weakly correlated when including all the specimens
(r ¼ 0.09523, P ¼ 0.014). Analyses of molecular
variance (AMOVA) offered consistent results
(Table 1). When comparing South America with
North America or with the Old World, there is a
higher percentage of variation between populations
than within populations, indicating that South
American populations are well differentiated. On
the other hand, when comparing North America and
the Old World the percentage of variation among
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populations is much lower, showing a higher degree
of similarity between them.
Scanning electron microscopy (s.e.m.)
There are significant differences in the diameter of
the spores of both ribotype groups (two-tailed Welsh
t-test, Po0.001), and also slight changes in the
ornamentation of their walls. Although both groups
have overlapping sizes, spores from ribotype group A
have in general bigger diameters (15.01 mm on average)
and, ignoring the outliers, a higher size range than
those from group B (12.69 mm on average) (Figures 2a
and b). Group A also has more variable wall
ornamentations, showing in most cases a very marked
reticulum and a polygonal shape, but also less
conspicuous ornamentation patterns were observed.
Spores from ribotype group B are usually round in
outline, with a generally less-marked reticulum.
Environmental niche models
Separate niche models were generated for each of
the two main groups of ribotypes. To predict species
occurrence over geographic space, we used a
maximum entropy model implemented in Maxent
(Phillips and Dudik 2008). Maxent was chosen
because it is a powerful tool in comparison with
other methods (Elith et al., 2006) even in the
presence of small data sets (Hernandez et al., 2006).
Predicted areas are located in warm arid territories
in both cases (Figure 3a). The map obtained for
ribotype group A predicts its presence in more
restricted areas, situated near the western coasts of
the continents. Ribotype group B has a broader
niche, which penetrates into more intra-continental
localities. In niche-overlapping areas from South
America, only samples from group A were collected
(hypergeometric distribution P ¼ 0.03) (Figure 3b),
and in those from North Africa both ribotype groups
were found. However, there is no information about
overlapping areas in North America.
Niche models of the ribotype groups are less
similar than random. Values of D and I indices
obtained from original data (0.339 and 0.597,
respectively) were smaller and out of the 95%
confidence interval (D: 0.69–0.72, I: 0.78–0.80) of
the distribution obtained from random pseudoreplicated models (Figure 3c).
Discussion
This work represents a first step in the study of
genetic variation in a complex of cryptic species of
myxomycetes in a geographical context. Our data
show geographically structured ribotypes, which are
congruent with the ME hypothesis. Implementing
the use of environmental niche models has revealed
that the two major ribotype groups have different
habitat preferences, although their models can
overlap in some areas. Models can also shed light
on many important unresolved biogeographical
questions, as the comparison of predicted areas
with actual presence data can be a powerful tool for
detecting the effect of barriers to dispersion. The
absence of specimens from ribotype group B in
South American niche-overlapping areas, which is
very unlikely only by chance (P ¼ 0.03), seems to
indicate that there have been impediments to its
dispersion to these areas. The future study of nicheoverlapping areas in North America from which
there is no information could confirm this pattern.
Geographic differentiation among ribotypes
Our results are consistent with data from mating
experiments in myxomycete cultures (El Hage et al.,
2000; Clark 2000; Clark and Stephenson 2000;
Irawan et al., 2000), which found that at least some
Group A
22
20
10 µm
18
Group B
16
14
12
Group A
Group B
Figure 2 Variability in spore morphology. A total of 32 specimens from ribotype groups A and B were randomly selected for
morphological studies. Ten spores per specimen were observed and measured (a): (s.e.m) pictures displaying one representative spore
from each studied specimen. (b): Box-and-whiskers plot of the diameter of the spores in mm.
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40
30
D
I
25
30
20
0.2705
15
0.5257
20
10
10
5
0.2
0.4
0.6
0.8
1
0.2
0.4
0.6
0.8
1
Figure 3 Environmental Niche Models. (a): Predictive ecological models based on the Maxent algorithm. Probabilities of presence 40.5
are represented for ribotype groups A (green) and B (red). Overlapping areas are shown in black. (b): Close-up of overlapping niche areas
in South America. Yellow dots denote localities where specimens from ribotype group A were found. (c): Niche comparisons of ribotype
groups A and B based on D and I as measures of niche overlap. Dotted lines represent niche overlap measures of the original data, and
bars show the expected degree of niche overlap when samples are drawn from the same distribution (i.e., pooled samples of occurrence
points from the ribotype groups).
widespread morphospecies may actually consist of
complexes of reproductively isolated clonal lines
that can be geographically restricted (Clark 2004).
Previous molecular studies on other myxomycete
morphospecies did not show any clear relationship
between genetic variants and the geographical origin
of the specimens (Winsett and Stephenson 2008;
Fiore-Donno et al., 2011). However, B. melanospora
displays a clear geographic pattern.
The structure found can be interpreted to understand the history of the species. As most basal clades
in the BI tree are Argentinean and Chilean populations, B. melanospora has its most probable origin in
South America. All North American specimens are
part of a well-supported clade that includes most
collections from the Old World but does not include
any sequence from Argentina or Chile. The TCS
analysis shows congruent results, with most closely
related sequences from ribotype group A and ribotype group B separated by nine missing ribotypes.
Mantel tests and AMOVA also support a North versus
South divergence in America, showing a correlation
between geographic and genetic distance in this
continent, and that, in addition, molecular variance
is lower in each subcontinent than in the total data
set considering all populations together. The
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morphological differences we found between ribotype groups support these conclusions. Although the
characters studied display overlapping ranges and
are not useful as diagnostic characters, they show
that genetic differentiation between the groups has
had some consequences for morphology.
Furthermore, our data do imply some long distance
migration. BI, TCS and AMOVA results suggest quite
strongly that North American populations were the
main source for populations from the Old World,
except for two groups of North African collections
and a highly divergent French specimen that were
more closely related to South American populations.
Another interesting result is that no ribotype was
found on both sides of the Andes – that is, in both Chile
and Argentina. The topology of the tree and the TCS
network do not show, however, two clearly defined
groups of populations from one side and the other. The
most likely explanation is that the Andes may have
acted as a semi-permeable barrier allowing multiple
colonization events across the mountain range.
Ribotype groups have different environmental niches
Environmental niche models provide evidence
for differences in the ecological preferences of
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B. melanospora ribotype groups, but there are
several areas where niches from both groups
overlap. Only specimens from ribotype group
A have been found in a niche-overlapping area
from South America that has been intensively
surveyed for myxomycetes (Lado et al., 2012).
This exclusion strongly suggests the existence of
barriers to dispersion at least between North
America and South America, and, unless there
is an effect of other unstudied environmental
factors, this is prima facie evidence for isolation
by geographic distance and inefficient global
dispersal. The presence of representatives of both
ribotype groups on native plants from the Old
World (Euphorbia, Pachypodium) and of group.
A individuals on North American plants
introduced in the Old World (Opuntia, Agave)
(see Supplementary Table 1) indicates that this
limited distribution may not be an artefact caused
by host plant specificity. One possible limitation
of the present analysis is that the modelled niches
take account of only climatic factors. Recent
studies (Aguilar and Lado 2012; Bates et al.,
2013) suggest that climate has a major influence
on the distribution of protists at medium to large
geographic scales. However, other potentially
important environmental variables such as
characteristics of the host plant, interaction with
other organisms, Ph, soil type, etc may also affect
the distribution patterns of B. melanospora.
Although the models reasonably predict the
distribution of the organisms, it is still possible
that a variable not included in the analyses is
causing the absence of ribotype group B from the
overlapping niche area.
Furthermore, the fact that one ribotype from group
B was found only once in Spain and many times in
Mexico and another was found once in the Canary
Islands and once in Ascension Island but many
times in Mexico shows that North American
ribotypes can survive in Europe or in ecologically
disparate Atlantic islands, suggesting that the
absence of most of them from these regions is
because of weak dispersal and not because of
ecological unsuitability.
It is important to note that there is no information
about the actual presence of B. melanospora in several
areas with high probability in the models – for
example, Southern Africa, Arabia and Australia. Most
of these areas have never been surveyed for myxomycetes, and this lack of data makes it harder to interpret
our results. It would be worthwhile searching for
B. melanospora in these areas; if it were not found, the
conclusion that B. melanospora has not been able
to reach all ecologically suitable areas during its
dispersive processes would be strengthened.
Human introductions
It is striking that Old World strains are nested within
the ancestrally New World groups A and B, but in
most cases forming a group of closely related
ribotypes in group B. AMOVA results also suggest
that in most cases Old World populations are more
similar to North American populations. This pattern
is most simply explained by multiple colonization
events from the Americas, especially from North
America, to Europe, oceanic islands, Africa and
Madagascar.
The four ribotypes found in Morocco do not
cluster together; one is almost identical to North
American sequences and one to Argentinean
sequences, whereas two are distinct Moroccan
ribotypes. Environmental niche models show that
there are areas in the north-western coast of Africa
where niches overlap and ribotypes of both groups
coexist. We suggest that there were four colonizations of Morocco from America, either directly or via
other unsampled localities in the Old World.
Assuming a similar rate of evolutionary change for
all strains, two Moroccan ribotypes would be so
recent as to be almost identical to their source
strains, and the other two would be more ancient –
long enough ago for separate ribotypes to have
evolved after colonization. Also, we postulate two to
three separate colonizations of Madagascar, all most
likely from North America. The two Brazilian
sequences are also most likely the result of introductions from North America. One of them is of the
same ribotype that was apparently introduced into
the Canary Islands and Madagascar.
Many specimens collected outside the Americas
were found on plants originally introduced from
North or Central America (Opuntia, Agave) (see
Supplementary Table 1), which quite strongly
supports the hypothesis that the dispersion of
B. melanospora has been facilitated by human
introductions of American succulent plants into
the Old World. If these strains had evolved in situ
one would expect them to be frequent on native Old
World plants.
If our interpretations are correct, then B. melanospora provides the first molecular evidence of
human introductions of a myxomycete, but it is
possible that at least six other morphospecies of
myxomycetes have been introduced in a similar way
from America to the Canary Islands (Lado et al
2007). Given the close similarity of most putatively
introduced strains, it is unlikely that our results are
misled by the absence of data from Africa due to an
incomplete sampling. Nonetheless, search for
B. melanospora in southern and east Africa is
an important test of our human introduction
hypothesis.
B. melanospora is probably a cryptic species complex
In the case of B. melanospora, samples clustered
forming six groups with a 97% similarity. Furthermore, group B constitutes a well-defined clade, with
high support and separated from other sequences by
a relatively long branch. This makes it likely that it
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is reproductively isolated from group A and forms
an independent evolutionary line. Groups A and B
are genetically, morphologically and geographically
strongly differentiated and it is unlikely that there is
much, if any, gene flow between them. Very likely
they are distinct biological species with different
geographic distributions. However, none of the
morphological characters studied allow an a priori
classification of the specimens in either group, and
therefore they cannot be considered as diagnostic
characters. It would not be surprising if the two
groups each include several cryptic biological
species.
Conclusions
Incorporating ecological aspects into biogeographical
studies of protists can make it possible to test the
EiE hypothesis. In cases where a serious lack of data
makes it difficult to carry out both ecological and
biogeographic studies, the use of environmental
niche models can be of great help, especially when
selecting the most appropriate variables for predicting an organism’s occurrence. One clear example is
myxomycete (mycetozoan) amoebozoans, which are
among the most widely distributed of all terrestrial
organisms, but also among the least known.
B. melanospora is a complex case in which
limited dispersion, isolation by distance, host
specificity and other ecological parameters have
acted, giving rise to a set of at least two cryptic
species with slight but not completely distinguishable morphologies. In addition, human introduction
of host plants may have played an important role in
facilitating multiple long distance colonization
events from the Americas to the Old World, as well
as two putative cases from Mexico to Brazil.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
We thank S L Stephenson, M Meyer, L H Cavalcanti and
R McHugh for contributing specimens and providing
comments on the species distribution and morphology,
and Fátima Durán, Guillermo Sanjuanbenito, Gemma
Andreu, Yolanda Ruiz and Juan Carlos Hernández for
technical work. This work was supported by Research
Projects CGL2011-22684/BOS and CGL2008-00720/BOS of
the Ministry of Science and Innovation of Spain.
Author contributions
MA, AMFD, CL and TCS designed the research; MA and
AMFD conducted the research; MA analysed the data; and
MA, AMFD, CL and TCS wrote the paper.
The ISME Journal
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