Population-Level Analyses Indirectly Reveal Cryptic Sex and

Life History Traits of Pseudoperkinsus tapetis (Ichthyosporea,

Opisthokonta): A Unicellular Relative of the Animals
Wyth L. Marshall*,1,2 and Mary L. Berbee1,2
1
Department of Botany, University of British Columbia, Vancouver, BC, Canada
2
Bamfield Marine Sciences Centre, Bamfield, BC, Canada
*Corresponding author: E-mail: WythMarshall@yahoo.ca.
Associate editor: Jeffrey Thorne

Abstract
Research article

We use population genetics to detect the molecular footprint of a sexual cycle, of a haploid vegetative state, and of lack of
host specificity in Pseudoperkinsus tapetis, a marine unicellular relative of the animals. Prior to this study, complete life
cycles were not known for any of the unicellular lineages sharing common ancestry with multicellular animals and fungi.
We established the first collection of conspecific cultures of any member from the unicellular opisthokont lineage
ichthyosporea, isolating 126 cultures of P. tapetis from guts of marine invertebrates ranging from clams to sea cucumbers.

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We sequenced fragments of the elongation factor alpha-like (EFL) and heat-shock protein 70 (HSP70) genes for a subset of
our isolates. Absence of heterozygotes from the EFL locus in 52 isolates provided evidence for haploidy. Phylogenetic
incongruence and a lack of support for linkage between two loci from 34 sequenced isolates signified a history of
recombination consistent with a sexual cycle. Shared haplotypes in different invertebrate species showed that P. tapetis
was not host specific. Based on estimates of the frequency of sex and on observations of cultures, we propose that P.
tapetis is transmitted between hosts via asexual endospores. New protists are continually being discovered, and, as this
study illustrates, analysis of culturable collections from natural habitats can transform a species from a near unknown to
a model system for better understanding the evolution of life histories.
Key words: effective population size, ichthyosporea, mesomycetozoa, protist, Pseudoperkinsus, recombination.

Introduction cycles have yet to be elucidated (Maldonado 2005; Carr

Traditionally, determination of microbial life cycles has re-
lied on tenacity, microscopy, and real-time observation.
Using population genetic techniques and samples of con-
specific isolates, we have worked in ‘‘reverse’’ describing as-
pects of an organism’s life cycle and ecology based on the
footprints left in DNA sequence variation.
During a survey of marine invertebrate digestive tracts
for novel and culturable unicellular osmotrophs, we cul-
tured 126 isolates of an ichthyosporean with 99.9% se-
quence similarity in the ribosomal small subunit rDNA
gene (SSU) to the shellfish symbiont Pseudoperkinsus tape-
tis (Figueras et al. 2000; Takishita et al. 2008). The ichthyo-
sporea are one of seven unicellular lineages belonging to
the eukaryote supergroup opisthokonta (Cavalier-Smith
1987; Adl et al. 2005). Prior to 1995, choanoflagellates were
the only known unicellular relatives of animals (Cavalier-
Smith 1987). Since that time, four additional lineages have
been discovered (Adl et al. 2005). Of these new additions,
the ichthyosporea are, so far, the most speciose (Mendoza
et al. 2002). Molecular phylogenetic analyses place the or-
igin of the ichthyosporea at the basal-most node on the
animal lineage after the animal/fungus divide (Ruiz-Trillo
et al. 2006, 2008; Steenkamp et al. 2006; Shalchian-Tabrizi
et al. 2008). For the unicellular relatives of animals including
choanoflagellates and ichthyosporea, ploidy and sexual
et al. 2009). Reasons for this include their recent discovery
and a lack of available cultures.
Our cultures of P. tapetis constitute one of the first col-
lections of conspecific isolates from any of the four new
lineages of unicellular metazoan relatives. Previously, P.
tapetis was cultured only twice, once from Japan and once
from Spain (Figueras et al. 2000; Takishita et al. 2008).
Rather than adapt cytological methods to a divergent
and possibly host-adapted organism, we applied popula-
tion-level genetic analyses to our collection of isolates.
Using a primer-based approach, we found two loci with
intraspecific variation, which we used to indirectly reveal
the ploidy, or chromosome copy number, of our isolates
and detect genetic evidence of recombination. Using meas-
ures of nucleotide diversity, we estimated the effective pop-
ulation size and frequency of recombination. We also
describe an enquiry into P. tapetis’s demographics and
its host specificity.
Genetic Evidence of Chromosome Copy Number
Ploidy is integral to a life cycle, but characterizing ploidy by
cytological observation can be difficult to adapt to new
groups of organisms. Flow cytometry could be used to sort
haploid from diploid cells (e.g., Bernander et al. 2001), except
that we had no means to establish baseline values for 1N and
2N chromatin amounts. A widely adaptable approach to

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2014 Mol. Biol. Evol. 27(9):2014–2026. 2010 doi:10.1093/molbev/msq078 Advance Access publication April 1, 2010
Cryptic Sex and Life History of Pseudoperkinsus · doi:10.1093/molbev/msq078
determining ploidy has been analysis of the frequency of het-
erozygotes in microsatellite data sampled from populations
(e.g., Oliveira et al. 1998; Brondani et al. 2000; Rynearson and
Armbrust 2000; van der Verde et al. 2001; Weeks et al. 2001).
The lack of heterozygotes from microsatellite data showed,
for example, that a population of Symbiodinium dinoflagel-
lates was haploid (Santos and Coffroth 2003). A similar prin-
ciple can be applied using sequence data from a population
with polymorphisms. Direct sequencing of polymerase chain
reaction (PCR) products from heterozygous diploids produ-
ces sequence chromatograms with double peaks at the seg-
regating sites (Clark 1990). The multicellular animals are
mainly diploid, and the higher fungi are mostly haploid or
dikaryotic. To assess chromosome copy numbers in P. tapetis,
we carefully screened the segregating sites of our sequence
chromatograms for double peaks, indicative of heterozy-
gotes and diploidy.
Genetic Evidence of Recombination in
Microeukaryotes
As in most microorganisms, asexuality is easily observed in
the few ichthyosporeans that grow in culture (Whisler
1968; Jostensen et al. 2002; Marshall et al. 2008). Meiosis
has yet to be reported in any ichthyosporean. Nuclear fu-
sion between coalesced uninucleate cells from adjacent fil-
aments, resembling the ladder-like conjugation of the green
alga Spirogyra, has been observed in the elongate ichthyo-
sporean Enteropogon sexuale (Hibbits 1978). However, the
fused cells did not grow or divide, and because E. sexuale is
host obligate, Hibbit’s observations were limited to short-
term study of dissected animals. Carr et al. (2008) argued
that the presence of long terminal repeat retrotransposons
in the genome of the choanoflagellate Monosiga brevicollis
indicated a sexual history because if the species had been
purely asexual, the retrotransposon build up would have led
to mutational meltdown and extinction. However, empirical
evidence showing a sexual cycle has yet to be uncovered for
any member of the unicellular relatives of animals.
Sexual recombination, while widespread, can be difficult
to observe and is unknown in many unicellular protists and
fungi (e.g., Reynolds 1993); however, the inability to find it
does not rule out its occurrence. On the other hand, even
when mating can be induced in the laboratory, it may not
necessarily occur in nature. Molecular analysis of variable
loci in populations has the advantage of being able to show
that sexual reproduction has occurred in nature by reveal-
ing patterns originating from cryptic recombination within
natural populations of pathogenic and free-living bacteria,
fungi, and protozoa (Maynard Smith et al. 1993; Burt et al.
1996; Geiser et al. 1998; LoBuglio and Taylor 2002; Xu 2004;
Campbell and Carter 2006; Cooper et al. 2007). Genomes of
asexual organisms are inherited as effectively identical cop-
ies of the parent. Sexual genomes are shuffled and reas-
sembled by independent assortment of chromosomes
and crossing over between paired chromosomes. We tested
whether P. tapetis had an incongruent phylogenetic history
between two loci, consistent with recombination. To the
opposite effect, we also tested for significant linkage, or as-
MBE
FIG. 1. Pseudoperkinsus tapetis isolation sites from Vancouver Island,
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British Columbia, Canada. Western sites were distributed within
Barkley Sound, near the Bamfield Marine Sciences Centre. East side
sites were from three beaches along a 3 km SSW shoreline of
Gabriola Island. Specific locations are listed in table S1.
sociation between these loci, indicative of either a clonal
population or close physical arrangement of the genes.
Life Cycle and Relationship with Host
Population-level analyses have been rare for the protozoa
and are lacking altogether for the unicellular opisthokont
lineages. Within the ichthyosporea, no genetic studies have
been conducted to look further at population structure or
host affinities. In order to use population-level genetic var-
iation to indirectly characterize the life cycle of P. tapetis,
we sequenced two protein-coding loci that exhibited intra-
specific variation. By screening sequences for heterozygos-
ity, we were able to determine the chromosome copy
number. With ploidy established, we were able to test
for evidence of a sexual cycle and estimate effective pop-
ulation size using measures of nucleotide diversity. Using
limited evidence of recombination, we were able to esti-
mate the frequency of sex by calculating the population
recombination rate based on the footprint left by homol-
ogous recombination in our sequences. In order to deter-
mine whether sex was obligate or facultative, we compared
the average time between recombination events with host
age. Ultimately, we combined these findings with culture
and isolation observations and proposed a life cycle that
included the host/symbiont relationship.
Materials and Methods
Animal Collection, Isolation of Strains and Culture
Conditions
We cultured P. tapetis from eight marine invertebrate host
collections plus one sample of water surrounding captive
oysters from the east and west sides of central Vancouver
Island, British Columbia, Canada, between August 2004 and
April 2007 (fig. 1 and table 1). Pseudoperkinsus tapetis was
not found from attempted isolations from an additional
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Marshall and Berbee · doi:10.1093/molbev/msq078 MBE
Table 1. Collection Location, Animal Host and Haplotype for the tion of a 150 ml aliquot of 0.5 l of water surrounding three
34 Sequenced Pseudoperkinsus tapetis Isolates. captive oysters. Ichthyosporean colonies were white and
Animal Host Haplotype raised and consisted of large round cells. We repeatedly
Location (number collected) Numbera Isolate IDb streaked each of the individual colonies from the starting
East 1 Mussel (5) 1* GM8 plates onto fresh media until they were axenic. For isolates
East 2 Mussel (5)c 1* MB4 CG2 and LE7, we observed asexual reproduction and stained
3 BM6, MB5, MB18
4 BM33
the nuclei with 4#,6-diamidino-2-phenylindole (DAPI) ac-
5* BM19 cording to Marshall et al. (2008). These strains are available
6 MB29 at the American Type Culture Collection (ATCC) under
12* MMB5, MMB3, MB13 accession numbers PRA-281/282.
13* BM17
14 MB16
Varnish clam (5)c 5* NO32
DNA Extraction, PCR Amplification, and
10 ON1M Sequencing
12* NO6, ON8, ON50M We extracted DNA from approximately 50–200 ll of cells
13* ON12 scraped from plates with confluent growth using a DNeasy
16 NO16 Plant Mini Kit (QIAGEN Inc., Mississauga, Ontario, Canada).
East 3 Manilla clam (5)d 2 PV3, PV38, VP2
3* PV25, VP7
We amplified and sequenced the SSU, internal transcribed
5* PV23 spacers ITS1 and ITS2 and the 5.8S ribosomal subunit rDNA

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11 VP19, VP17
West 1 Pacific oyster (3)c 7 CG2
8 CG5
15 CRG9
West 2 Chiton (3) 9 KS1
West 3 Sea cucumber (5) 17 LE7
West 4 Oyster water 18 OI22B
NOTE.—East 1–3 refers to each of three beaches on Gabriola Island, and west 1–4
corresponds to four sites within Barkley Sound. Specific locations are provided in
table S1.
a temperature of 48 °C and then cloned the fragment into the
Asterisks mark haplotypes that were found in more than one host collection.
b
A representative of each haplotype per host collection was included in the
‘‘individual’’ data set (see figs. 2 and 3).
c
Animal collections where the number of haplotypes is equal to or greater than
the number of animals pooled.
d
The number of haplotypes is at least 5 (see table S1).
32 animal collections (supplementary table S1, Supplemen-
tary Material online). Because the geographic extent of
populations of ichthyosporeans is unknown and incorrect
estimations of population size can sometimes give false in-
dications of clonality (e.g., Campbell et al. 2005), we sam-
pled animals from overlapping (cohabiting) sites as well as
beaches separated by 1–200 km of coastline (fig. 1, table 1,
and supplementary table S1, Supplementary Material on-
line). Animals from the east side were collected from three
beaches, each separated by 1 km along the southern shore
of Gabriola Island. Animals from the west side were col-
lected from four sites within Barkley Sound, each separated
between 0.75 and 2.5 km. Specific locations are provided in
supplementary table S1, Supplementary Material online. To
obtain culture material, we pooled portions of the digestive
tracts from three to five animals from each of the eight host
collections (Marshall et al. 2008). For most animals, we used
pieces from all gut regions; for bivalves, we used a portion
of the digestive gland and syringed stomach material. We
diluted the gut contents in 400 ll of sterile seawater and
inoculated culture plates with 10 and 30 ll of the slurry
(Marshall et al. 2008). As a control, we cultured undiluted
coelomic fluid (non-bivalve animals) and mantle cavity fluid
(bivalves) from five of eight pooled animal collections. We
also isolated cultures from a pellet formed from centrifuga-
gene (ITS), and a region of the elongation factor alpha-like
gene (EFL) directly from PCR products (Marshall et al. 2008),
except that we used internal primers ELF3 and ELR3 (sup-
plementary table S2, Supplementary Material online) to se-
quence the EFL. For initial primer design, we amplified
a region of the cytosolic heat-shock protein 70 gene
(HSP70) using primers H7150F and H7965R (supplementary
table S2, Supplementary Material online) with an annealing
vector pCR2.1 using the TOPO TA cloning kit (Invitrogen)
and sequenced clones. We then amplified the HSP70 from
our isolates using our new internal primers H7F2 and H7R2
(supplementary table S2, Supplementary Material online)
with an annealing temperature of 52 °C and sequenced
the PCR products directly. Sequences were determined
by Macrogen at Seoul, Korea, or the Nucleic Acid Protein
Service Unit at the University of British Columbia, Canada.
To verify point mutations, we sequenced each allele from at
least two PCR products and in both directions (supplemen-
tary table S1, Supplementary Material online), mixing all
PCR reagents in a laminar flow hood to reduce or eliminate
cross contamination. As a check, we reamplified and rese-
quenced 36% of HSP70 and 50% of EFL genes (fig. 2 and sup-
plementary table S1, Supplementary Material online) and
found no evidence for cross contamination. The haplotypes
represented by isolates GM8, BM19, VP19, PV23, LE7, OI22B,
BM6, PV25, NO6, MMB5, ON1M, PV3, BM17, MB29, and
ON12 were verified either through being reamplified and
resequenced or by being found in other isolates (supple-
mentary table S1, Supplementary Material online).
We worked under the assumption that conspecific iso-
lates could be recognized and identified as P. tapetis based
on a phylogenetic species concept. All P. tapetis isolates
formed a monophyletic group. A subset of eight of our iso-
lates (supplementary table S1, Supplementary Material on-
line) were 100% identical in SSU sequence to each other
over a maximum overlap of 1718 bp and were 99.9%
identical to the only two other known P. tapetis isolates
(Figueras et al. 2000; Takishita et al. 2008). All 126 of
our isolates shared 99–100% ITS identity to each other

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Cryptic Sex and Life History of Pseudoperkinsus · doi:10.1093/molbev/msq078
FIG. 2. The genealogical network from the HSP70 locus (top)
conflicts with the network from the EFL locus (bottom), suggesting
a history of recombination between the two loci. Each alphanu-
meric code designates an individual isolate; isolates collected from
the west side are identified by italics (for isolate details, see fig. 3 and
table 1). Code color specifies membership in one of three HSP70
haplotype networks, designated in blue, black, and red. Instead of
the expected pattern in the absence of recombination, where
isolates of the same color would generally group together in both
the HSP70 and the EFL networks, the rearrangement of the color
patterns in the EFL network suggests recombination. Networks were
generated by TCS (Clement et al. 2000). Numbers above branches
are branch lengths from TCS; numbers below are parsimony
bootstrap percentages from PAUP* (Swofford 1998). Consistency
index (C.I.), Retention Index (R.I.), segregating sites (S), and tree
length (L) are shown for both trees. Dashed lines between the three
HSP70 networks show hypothetical connectivity after the TCS
connection limit was increased to 60 steps. This diagram also
illustrates three alternative approaches to designating alleles for
further analysis. Circles around isolates with identical sequences
designate 11 HSP70 and 10 EFL alleles. To minimize the effects of
recent somatic mutations, three alternative approaches to desig-
nating alleles were used: 1) All sites were considered (circles 1–10 or
11), 2) sequences with only one unique difference were lumped
together as the same allele (A–F or H, dashed boxes), or 3) alleles
were defined using only balanced sites (fig. 3) (solid boxes E/H1–5).
and to the single isolate of P. tapetis from Spain over a re-
gion with a maximum overlap of 667 bp including ambig-
uous sites with secondary peaks. Compared with the P.
tapetis isolate from Japan, our isolates shared 96.9–98.8%
ITS similarity over a maximum overlap of 675 bp. In con-
trast, other ichthyosporeans that we isolated (data not
shown) were not considered to be conspecific with P. tapetis
MBE
and were excluded from this study because they were at
most 88.4% identical to P. tapetis over a 725-bp region of
overlap in their ITS regions. These levels of identity were
comparable with the 5.1% interspecific versus 0.51% intra-
specific ITS divergence within a data set of Penicillium
species (Seifert and Samson 2007). Of our 126 putatively
conspecific isolates, we chose 34 isolates for sequencing
of the EFL and HSP70 (table 1). These isolates represented
a selection from all eight animal hosts (table 1 and supple-
mentary table S1, Supplementary Material online). Because
inbreeding can result in elevated levels of homozygosity, we
increased the number of sequences from the EFL locus to 52.
Genetic Analysis
We aligned sequences in ClustalX v1.8 (Thompson et al.
1997) and edited them in MacClade 4.08 (Maddison WP
and Maddison DR 2000). We calculated pairwise nucleotide
diversity, p, and theta per site, hw (Watterson 1975), in
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DnaSP v.4.50.3 (Rozas et al. 2003) based on a data set of
24 individuals (defined below). To construct parsimony
trees, we used TCS v.1.13 (Clement et al. 2000) and PAUP*
v.4.0b10 (Swofford 1998). Parsimony bootstrapping in
PAUP* involved 500 heuristic search replicates with 10 ran-
dom sequence additions with tree-bisection reconnection
(TBR) branch swapping limited to 106 rearrangements. We
used the most parsimonious tree from each locus to cal-
culate the consistency index (CI), retention index (RI), and
tree length using PAUP*.
To decide chromosome copy number, we checked orig-
inal chromatograms for each of the EFL sequences, looking
for double peaks at any of the 19 segregating sites identified
after aligning all 52 EFL sequences.
Definition of Data Sets
In order to eliminate clone siblings that may have been iso-
lated during dissection and culturing, we included only
unique haplotypes from each animal collection (hereafter
referred to as ‘‘individuals’’) in our first data set (n 5 24).
Asexual reproduction was obvious in culture. For analyses
where a history of sexual recombination could be obscured
by including asexual siblings, we defined two additional
data sets with varying degrees of correction to eliminate
possible clones. Some isolates came from animals collected
from the same beach. Assuming clones dispersed locally, we
used unique haplotypes from the each location for a second
‘‘location-corrected’’ data set (n 5 21). In order to assess
the relationship of the loci independent of population
structure, we used a third data set consisting of unique
haplotypes (n 5 18). In addition to allowing for alternative
degrees of stringency in removing possible clones, we also
applied three alternative definitions of alleles for analyses
where results depended on how stringently the alleles are
defined (see fig. 2).
Expected Number of Individuals
Animals collected from the east side of Vancouver Island
gave rise to a high number of isolates. When isolates with
identical haplotypes were cultured from the same animal
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Marshall and Berbee · doi:10.1093/molbev/msq078 MBE

FIG. 3. Polymorphic sites from the amplified regions of the EFL and HSP70 loci shown for each of 24 individuals or unique haplotypes per host
collection. Each individual was assigned 1 of 18 unique haplotypes defined after the EFL and HSP70 sequences were concatenated. Horizontal lines
separate isolates based on their combined haplotype designation. A small ‘‘g’’ denotes a gap, and each gap was coded as one character regardless of
its length. The noncoding region of the HSP70 is shaded in grey. The polymorphic region used to calculate the expected heterozygosity of the EFL is
outlined by a box. Balanced sites were defined as any site where polymorphisms were present in at least three individuals and are marked by an
asterisk above the consensus line. Allele and haplotype numbers correspond to figure 2. Specific collection locations are listed in table S1.

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collection, it was impossible to know whether they origi-
nated from one or more initial infections of the host. How-
ever, assuming that haplotypes infected randomly and that
infections were Poisson distributed based on average hap-
lotype frequency, we used an arithmetic approach to esti-
mate a fourth data set, the actual or ‘‘expected’’ number of
infective individuals based on our sequenced ‘‘individual’’
isolates from the east side. Where f was the number of hosts
infected with the same haplotype, f/4 was the observed fre-
quency of finding the haplotype among our four host col-
lections,
ln (1
f/4) was the Poisson-corrected expected
frequency of the haplotype infection of four host collec-
tions, and finally (
ln (1
f/4)*4) was the number of times
that the same haplotype of P. tapetis would have been ex-
pected to independently infect the pool of four hosts. If, for
example, the same haplotype of P. tapetis was isolated from
two of the four hosts, its observed frequency was 0.5, its
expected frequency was 0.69, and the expected number
of cells independently initiating infection was 2.77.
Tests for Evidence of Clonality and Recombination
Genetic Tests for Clonality
Linkage disequilibrium or nonrandom association among
alleles between loci can arise from genetic isolation, in-
breeding or lack of recombination, and clonal expansion.
We tested the null hypothesis of recombination by calcu-
lating the index of association (IA) (Brown et al. 1980; May-
nard Smith et al. 1993) using Multilocus v.1.3 (Agapow and
Burt 2001). Values range between 0 for randomly associ-
ated alleles between loci (due to panmictic mating) and
1 for complete linkage. To assess significance of the alter-
nate hypothesis of clonality (P , 0.05), we compared the
observed data set with 10,000 replications that mimicked
the effects of random mating. The IA was calculated and
tested for all three allele definitions (fig. 2) for the individ-
ual, location corrected, and unique haplotype data sets (ta-
ble 2). We also calculated and tested the IA for the
individuals and unique haplotypes on the east side (table
2).
The parsimony tree length permutation test (PTLPT)
(Archie 1989; Burt et al. 1996) uses all the sequence data
and therefore takes into account similarity among alleles
as well as allele frequency. For the PTLPT in the absence
of recombination, the combined tree length for both loci
should be significantly shorter (P , 0.05) than trees gener-
ated after simulating random mating with random mixing of
the alleles. We generated 1,000 randomizations using Multi-
locus with alleles coded as character states for each locus for
the individual and unique haplotype data sets. Following
randomization, we replaced the allele codes with the orig-
inal sequence data and calculated the tree lengths in PAUP*
according to the specifications provided by Multilocus.
Tests for Recombination
Sexual recombination in eukaryotes is a consequence of
segregation and independent assortment of chromosomes
during meiosis resulting in different genealogies for loci on

Table 2. Significant (P , 0.05) Indexes of Association (IA) Between HSP70 and EFL Alleles Were Evident When All Individual Isolates Were
Included, But Not When Data Sets Consisted of Unique Haplotypes or Location Corrected Haplotypes.
Allele Definitions Isolates Included Number of Isolates IA P
All sites considered All individuals 24 0.1814 0.015
Alleles reduced by one variable site (fig. 2) All individuals 24 0.1482 0.025
Alleles defined by balanced sites (fig. 2) All individuals 24 0.1328 0.036
All sites considered Location corrected 21 0.0717 0.23
All sites considered Unique haplotypes 18 20.0736 1.0
All sites considered East side individuals 18 0.1949 0.034
All sites considered East side unique haplotypes 12 20.1277 1.0

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Cryptic Sex and Life History of Pseudoperkinsus · doi:10.1093/molbev/msq078
separate chromosomes. We tested for incongruent histories
between the HSP70 and the EFL using the incongruence length
difference (ILD) test (Farris et al. 1994) for the data set of 24
individuals. The ILD tests a null hypothesis of clonality by com-
paring the summed tree length of the observed data with the
sums after random partitioning. We generated 1,000 trees in
PAUP* using heuristic searches with random sequence addi-
tion and TBR branch swapping limited to 106 rearrangements.
Our cut-off for significance was P , 0.01, and we excluded in-
variant characters (Cunningham 1997).
To map the history of recombination between the
two loci, we made recombination networks (Huson and
Kloepper 2005) for the EFL, HSP70, and both regions com-
bined in Splits Tree4 v4.10 using the data set of 24 individ-
uals (Huson 1998; Huson and Bryant 2006). To minimize
homoplasies and keep the number of segregating sites
within each locus comparable, we only used sequence from
the second exon of HSP70 (fig. 3) when the loci were com-
bined. We tested for recombination within each locus and
within the combined loci using the ‘‘pairwise homoplasy in-
dex’’ (PHI) (Bruen et al. 2006) implemented in Splits Tree4.
Homologous recombination is a second consequence of
meiosis and measurements of covariation between nucleo-
tides provides a basis for estimating the frequency of sexual
recombination. We tested both loci using the PHI test
(as above). We also compared alternate methods, likeli-
hood analysis of recombination in DNA (LARD) (Holmes
et al. 1999), and Bootscan (Martin, Posada, et al. 2005), im-
plemented by RDP3 software (Martin, Williamson, et al.
2005) after excluding alleles with single unique mutations.
To measure the degree of covariation of nucleotides at dif-
ferent sites within our 24 individuals, we calculated the
population recombination parameter (q) from the inter-
genic region of the HSP70 locus using the moment method
of Hudson (1987) and R minimum of Hudson and Kaplan
(1985) in DnaSP. We also used LDhat v.2.1 (McVean et al.
2002) to calculate q using Wakeley’s moment method
(Wakeley 1997) and the pairwise approximate likelihood
method (McVean et al. 2002).
Effective Population Size
We calculated the effective population size (Ne) according
to Ne 5 hw/2l. The upper and lower bounds of the mu-
tation rate per base pair per mitosis (l) were 0.22–2.5 
10
9 based on ranges from unicellular eukaryotes (Lynch
2006). We calculated overall per site nucleotide variation
(hw) from the HSP70 intron and the 4-fold degenerate sites
from the coding regions of HSP70 and EFL for all 24 indi-
viduals. For comparison, we also calculated hw and Ne for
the EFL using the east and west side isolates separately.
Frequency of Sex
We estimated the frequency of sex by comparing the rel-
ative contributions of same site variation due to mutation
and covariation of nucleotides due to recombination to
overall nucleotide diversity (Tsai et al. 2008). In the stan-
dard neutral model, Ne can be calculated from the popu-
lation mutation parameters (as above) or recombination
parameters according to Ne 5 q/2(rr) (Tsai et al. 2008).
MBE
The population recombination parameter (q) is the mea-
sure of overall covariation of nucleotides at different sites,
and (rr) is a compound value based on the observed num-
ber of chiasmata in Morgans per kilobase (M/kb) during
meiotic division or per meiosis recombination rate (r) mul-
tiplied by the frequency of sex (r). We used the HSP70 intron
to calculate hw to avoid the effects of selection and codon use
bias. We estimated the per meiosis recombination rate (r) as
being between 0.000017 and 0.0048 M/kb based on obser-
vations from unicellular eukaryotes (Awadella 2003; Lynch
2006). An r value of 10
3 M/kb corresponds to roughly 1
chiasma for a 1 Mb chromosome. We did not account for
inbreeding nor consider the series of mitotic divisions be-
tween endospore growth and release of new daughter cells.
Because we had so few isolates from our west side sites, we
were unable to confidently test for population connectivity.
However, alleles from both loci were shared by isolates from
both sides of the island, and we assumed that our 24 indi-
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viduals were part of a single population.
Results
ITS Variation
Of a total of 975 sequenced sites within the ITS, 29 were
frequently ambiguous, with secondary peaks clearly visible
within cleanly sequenced regions. However, resolution and
strength of the second peaks at the polymorphic sites were
inconsistent between sequence reads from single isolates.
In most eukaryotes, ribosomal regions including the ITS are
present in multiple tandem repeats. The patterns of se-
quence polymorphism in the ITS were consistent with var-
iation among repeats rather than allelic variation, and the
ITS variants were not analyzed further. When we ignored
ambiguous sites, 123 isolates (including CG2) were identi-
cal and 3 others, including LE7 and OI22B, differed at three
sites. A single locus such as the ITS is not sufficient to define
a species, and our population could still have included hy-
brids or more than one species. Isolates LE7 and OI22B were
the most divergent in their ITS, EFL, and HSP70 sequences.
We repeated tests for index of association, PTLPT, and ILD
without LE7 and OI22B, but the significance of our results
was unchanged.
Population Variation and Lack of Heterozygosity
The EFL data matrix, based on the 24 individual isolates,
had 922 nucleotides with 19 segregating sites and 10 alleles
(figs. 2 and 3). The amino acid translation was 307 codons
in length, and all but one nonsynonymous substitution
were in the first or second position. All synonymous sub-
stitutions were in the third position. The HSP70 matrix,
based on 24 individuals, consisted of 1,006 nucleotides with
64 segregating sites and 11 alleles (figs. 2 and 3). Two coding
regions with a total of 13 synonymous substitutions flanked
a 524 bp intron (fig. 3).
If our isolates were diploid, double peaks indicative of
heterozygotes should have been evident as conflicting sig-
nals on DNA sequence chromatograms. For example, as-
suming Hardy–Weinberg equilibrium, the frequency of
2019
Marshall and Berbee · doi:10.1093/molbev/msq078 MBE
heterozygotes at a chosen site within the EFL (fig. 3) should
have been 56.9% based on the frequencies of the alleles in
the individual data set. However, the observed frequency of
heterozygotes among the 52 isolates sequenced for the EFL
was zero. There were also no double peaks at any of the
polymorphic sites within the 34 isolates sequenced for
the HSP70. We therefore conducted all further analyses un-
der the premise that P. tapetis was haploid.
We found six unique haplotypes, one time each, from the
west side of Vancouver Island, and all the other isolates came
from four host collections from three beaches within a 2 km
range on the east side of the island (table 1 and fig. 1). The 12
different haplotypes found on the east side represented
a minimum of 18 independent host colonizations. The num-
ber was a minimum because it was calculated by ignoring
repeated cultures with the same haplotype from the same
host collection under the assumption that genetically iden-
tical cells originated and spread from a single initial coloni-

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zation. However, in some instances, cells of the same
haplotype may have colonized independently. Factoring
in the probability of multiple independent infections by
the same haplotype increased the possible number of inde-
pendent colonizations in the east side hosts from 18 to 25.
Haplotypes were not correlated with host species. In-
stead, the same haplotypes were found in more than
one host species (table 1). An excess of P. tapetis haplotypes
relative to number of host animals pooled for culture in-
dicated that some host animals contained more than one
haplotype (table 1). One host sample of five animals had
eight different P. tapetis haplotypes, and several animal col-
lections had a number of haplotypes equal to the number
of animals sampled (table 1).

Evidence of Asexual Reproduction and Clonality

Morphological and Genetic Evidence of Clonality
Uninucleate endospores grew into multinucleate plasmodia
before maturation (fig. 4a). DAPI-stained cells undergoing
asexual reproduction in culture typically had 32, 64, or more
nuclei by the time endospores were visible. Numbers higher
than 64 nuclei per cell could not be accurately counted. The
rare cells that had nuclear counts on the order of 250 were
often irregular in shape and did not appear to have endo-
spores. Nuclear division was synchronous, and nuclei in ma-
ture cells were the product of five to nine mitotic divisions.
After 3–5 days, endospores were usually released through
pores or splitting of the parent cell wall (fig. 4b).
The population-level footprint of clonal or asexual re-
production was evident as a significant IA value when all
individuals were included (table 2). The IA decreased as al-
lele definitions were adjusted to encompass minor variants
but still indicated significant clonality (table 2). However,
the null hypothesis of recombination could not be rejected
when the sample was reduced to the location-corrected
data set (table 2) or the third data set that consisted of
unique haplotypes for both sides or from the east side
alone (table 2). Similarly, when sequences were used in
place of allele codes (PTLPT), the observed tree length
was significantly shorter (P , 0.023) compared with a per-
FIG. 4. Asexual reproduction in Pseudoperkinsus tapetis. Like most
ichthyosporeans, P. tapetis produces a multinucleate parent cell,
followed by the release of asexually derived endospores. (a) (i–vi)
DAPI stain of nuclear proliferation in P. tapetis. Cells start as
uninucleate endospores, nuclei divide synchronously producing 2, 4,
8, 16, and so forth, multinucleate stages. Scale bar equals 20 microns.
(b and c) Uninucleate nonmotile endospores are released (b) when
the parent cell wall splits into two valves or (c) by squeezing through
pores in the parent cell wall. Scale bars equal 20 and 10 microns.
mutated data set including all individuals; however, the null
hypothesis of recombination could not be rejected using
the data set consisting of unique haplotypes (P . 0.078).
Recombination, Effective Population Size, and
Frequency of Sex
Genetic Evidence for Sex
Interlocus phylogenetic conflict pointed to a history of re-
combination in the population. The TCS parsimony net-
works from each locus (fig. 2) were incongruent, and

2020
Cryptic Sex and Life History of Pseudoperkinsus · doi:10.1093/molbev/msq078 MBE

FIG. 5. Inter- and intralocus recombination reconstructed using Splits Tree4. Reticulations representing potential recombination events are

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shown in bold grey. Networks are not drawn to scale. (a) Networks from the combined EFL plus the HSP70 exon 2 region, compared with the
exon 2 region of HSP70 region alone (inset). The dashed lines represents reticulations shared by both trees. (b) Recombination network using
all 1,006 base pairs from our HSP70 sequence.

the tree lengths of the combined data sets were not addi- the east side and the intergenic region of the HSP70 had
tive. The C.I., R.I., and lengths calculated in PAUP* for each higher diversity than the degenerate sites from the coding
locus were 0.94, 0.99, and 68 for the HSP70 and 1.0, 1.0, and regions (table 4). Using intergenic and degenerate sites, the
20 for the EFL, respectively. The values for the combined Ne was on the order of 106 to 107 depending on the mutation
data sets were C.I. 5 0.8077, R.I. 5 0.9373, and length 5 rate (table 4).
104. When the two loci were analyzed separately, the ob- Frequency of Sex
served number of steps exceeded the minimum number of Ratios of sexual to asexual generations or frequency of sex,
steps only by 1 (EFL) or 4 (HSP70), but when the loci were r, ranged between 1 sexual cycle for 154–495,000 asexual or
combined, the tree was 21 steps longer than the minimum mitotic cycles with a median value of approximately once
and the ILD test was significant (P 5 0.001). Similarly, the for every 22,700 asexual cycles when q equalled 1 (fig. 6).
combined network for the coding region of the HSP70 and Median values were based on the two less extreme esti-
the EFL had several reticulations (fig. 5a), suggestive of re- mates. Our estimate of q was based on a relatively short
combination. A reticulation was detected when a fragment fragment of DNA, and q can vary dramatically between loci
of the second exon of the HSP70 was analyzed alone (fig. (Awadella 2003) for reasons including chromosomal posi-
5a), although without significant support from the PHI test tion. When we repeated our calculations using a hypothet-
(P 5 0.2), whereas the EFL had no reticulations. The PHI ical high q of 50, the frequency of sex ranged between 1 in 3
test for the combined data was significant (P , 0.01). and 1 in 9,910 asexual generations, with a median value of 1
Reticulations and intralocus recombination were evi-
dent in the entire sequenced region of the HSP70 (PHI:
P , 0.01) (fig. 5b). Both Bootscan and LARD detected three
significant recombination events (P  0.05) but neither Table 3. Estimates of Effective Population Size Times the
could confidently distinguish daughter sequences from Frequency of Sex (Ner)a Based on Several Estimates of Population
the minor parent or identify breakpoints. Both methods Recombination Parameter qb Calculated for the Data Set
suggested OI22B as a major parent, BM6 as a minor parent, Including All 24 Individuals.
and NO6 and CRG9 were two potential daughters. A third Effective Population Size 3
scenario was BM6 as a major parent, OI22B as a minor Frequency of Sex (Nes)a
parent, and LE7 a daughter. Method rb r/uw rb 5 0.000017 rb 5 0.0048
Effective Population Size Using hw Wakeley (1997) 0.285 0.0136 8,380 30
Rm/pairwise 1 0.044 29,400 104
Estimates of q for the HSP70 ranged between 0.285 and 1.6 Hudson (1987) 1.6 0.070 47,100 167
(table 3). Estimates of nucleotide diversity (p) and hw were
NOTE.—The q/hwc ratio was calculated using hw from the HSP70 intron and Ner
higher for the HSP70 than the EFL, although this difference based the upper and lower bounds of per meiosis recombination rate, rb.
was minimized when only 4-fold degenerate sites were con- a
(Ner) is the effective population size, Ne, times the frequency of sex, r. (Ner) is
sidered (data not shown). hw ranged between 0.0126 and calculated based on Ner 5 q/2r.
b
q and r are expressed in Morgans per kilobase (M/kb).
0.0227 using degenerate or intergenic sites (table 4). For c
hw is Watterson’s (1975) estimator of nucleotide diversity expressed per
the EFL, nucleotide diversity was higher on the west side than kilobase.

2021
Marshall and Berbee · doi:10.1093/molbev/msq078
Table 4. Effective Population Sizes (Ne) Based on Intergenic and 4-fold Degenerate Sites Given an Upper and Lower Bound for Mutation Rate l.
Locus and Sites Used (number of nucleotides) uw
HSP70 coding (74) 0.0179
HSP70 intron (524) 0.0227
EFL coding, both sides (155) 0.0126
EFL coding, east side Vancouver Island (155) 0.0110
EFL coding, west side Vancouver Island (155) 0.0176
NOTE.—Nucleotide diversity (hw) was calculated from all 24 individuals.
sexual generation for 418 asexual generations. A data set
based on the expected number of infective individuals
as estimated with a Poisson correction for repeated infec-
tions by the same haplotype increased the number of in-
dividuals by 7 to 31. Because the expected data set included
a higher proportion of identical isolates, the resulting esti-
mates of the frequency of sex were slightly lower, with a me-
dian value near 1 sexual cycle for 23,900 asexual cycles
when q equalled 1.
Discussion
The combined results from microscopy and genetic anal-
ysis from our collection of conspecifics enabled us to indi-
rectly unveil aspects of the life cycle and ecology of P.
FIG. 6. Range of possible frequencies of sexual reproduction in
Pseudoperkinsus tapetis, given three estimates for the population
recombination factor (q) calculated for the heat-shock protein 70
locus (x axis) where a y axis value of 1 is equivalent to no asexual
reproduction. The vertical ranges represent alternative assumptions
about the per replication recombination rate (r) and mutation rate
(l). Black symbols represent l 5 2.5  10
9 and white symbols
l 5 2.2  10
10, and rectangles represent r 5 4.8  10
3 and
circles r 5 1.7  10
5. Median values (*) were calculated between
the two least extreme mutation and recombination rate combina-
tions. If sexual reproduction was less frequent than once in 700
nuclear divisions (shaded region), and assuming that P. tapetis is
retained in its host for a year, then initial host infection is probably
via asexual spores. The dotted lines are cut-offs, below which asexual
spores would constitute the infective stage given retention times or
host lifespans of 1, 5, and 10 years. Ninety percent inbreeding would
increase the range of frequency of sex by a factor of ;5.
2022
MBE
Ne Given m 5 0.22 3 1029 Ne Given m 5 2.5 3 1029
40,680,000 3,580,000
51,590,000 4,540,000
29,090,000 2,560,000
25,000,000 2,200,000
31,360,000 2,760,000
tapetis that could not be observed in the field or in culture.
The complete absence of heterozygotes at polymorphic
sites suggested that our P. tapetis population lives in its in-
vertebrate host primarily as a haploid and that it has a hap-
loid asexual cycle. If life cycles in other ichthyosporea are
predominantly haploid, Hibbits’ (1978) observations of
cells of E. sexuale undergoing nuclear fusion could be inter-
preted as fusing haploid cells in the process of forming a zy-
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gote. No diploid asexual cycle was evident in P. tapetis,
which may indicate that it has a short-lived zygote, or
a patchy distribution of haploid and diploid populations,
or that diploids for some reason do not colonize animals
or grow in culture.
An alternate explanation for a lack of observed hetero-
zygotes could be a diploid population that is highly inbred.
Johnson et al. (2003) found only one heterozygote among
28 isolates from a recombining population of the diploid
terrestrial yeast Saccharomyces paradoxus. For P. tapetis,
haploidy seems more likely than absolute inbreeding. Con-
sidering the large number of alleles, outcrossing would
have to be extremely rare to maintain 100% homozygosity.
No forms of cellular fusion such as those seen in yeasts
(Johannsen and van der Walt 1980; Johnson et al. 2003) were
observed under our culture conditions. Compared with
nonmotile terrestrial microorganisms, P. tapetis should have
more opportunities to disperse because cells are carried in
currents and tides and to outcross because host animals in-
gest cells and concentrate them. Based either on the min-
imum number of colonizations or on the calculated
expected number of colonizations, the number of infections
was greater than the number of animals pooled in several
samples. Because P. tapetis was not genetically isolated
within its host, it should have potential partners for
outcrossing.
Evidence for Both Clonality and Recombination
Tests for clonality, such as the index of association, use pan-
mictic mating as a null hypothesis and showed that pan-
mixis alone was not an adequate explanation for genetic
structure. Additional evidence in favor of clonality was that
all four gametic types were never found for any pair of al-
leles (Tibayrenc et al. 1990), but the absence of some of the
expected recombinant combinations may have been due
to insufficient sampling. The lines of evidence for sexuality
relied on the premise that under a clonal model, all regions
of the genome have the same history. Once our data were
reduced to unique haplotypes, the history of meiotic rear-
rangement was evident, and from the index of association,
Cryptic Sex and Life History of Pseudoperkinsus · doi:10.1093/molbev/msq078
we were unable to reject a null hypothesis of recombina-
tion. The EFL and HSP70 had significantly incongruent his-
tories, an expected consequence of a meiotic stage with
independent chromosome assortment (or recombination
between loci on the same chromosome). Evidence of intra-
locus recombination in the HSP70 provided more positive
support for a sexual cycle in our haploid organism where
meiosis would have offered the only opportunity for cross-
over between different alleles from a single copy homolo-
gous gene. We did not find intralocus recombination in the
EFL, but this was unsurprising as intralocus recombination
is less common than interlocus recombination and the
EFL was less variable than the HSP70 (fig. 3), so the prob-
ability of detecting recombination within the EFL was
lower. Although difficult to observe directly, the evidence
for recombination was not unexpected. Meiosis is likely an
integral process in all extant eukaryotes except for some
lineages with recent losses (Ramesh et al. 2005).
The q/hw ratio was lower in P. tapetis (table 3) than in
S. paradoxus (0.31–0.81) (Tsai et al. 2008) or Plasmodium
falciparum (0–6.2) (Awadella 2003) but greater than that
in Trypanosoma cruzi, a predominantly clonal species
(Llewellyn et al. 2009) with a q/hw of zero (Awadella
2003). In the animals Drosophila melanogaster and Caeno-
rhabditis remanei, the ratios ranged from 0.6 to 7.6 (Tsai
et al. 2008). Fraser et al. (2007) estimated that q/hw ratios
greater than 0.25 prevented clonal divergence in bacteria,
which rely on homologous recombination for sex. Uniformly
low values, below 0.07 in three different methods of
analysis, suggested that clonality has been an important force
in shaping the genetic structure in our P. tapetis population.
This superimposition of clonality on sexuality is often ob-
served in populations of unicells (e.g., Ngouanesavanh
et al. 2006; Bui et al. 2008).
Frequency of Sex
Our estimates of the ratio of sexual to asexual cycles in P.
tapetis were based on a series of assumptions. One assump-
tion was that no inbreeding was occurring. However, if in-
breeding does occur, as is likely the case, our calculations
will underestimate mating frequency. The higher the fre-
quency of inbreeding, the larger our underestimate of mat-
ing frequency would be. Inbreeding coefficients are
unknown for P. tapetis but using Ne 5 q/2r (1
F) and
Ne 5 h (1 þ F)/2l where F is the inbreeding coefficient (Tsai
et al. 2008), assuming that F 5 0.5 would increase the q/hw
ratios and mating frequency estimates by ;1.33 times,
whereas F 5 0.9 would increase them by ;5 times.
Our calculations of the frequency of mating are based on
the relative frequency of meiotic compared with mitotic
divisions, equating a nuclear mitotic cycle to an asexual
generation. We could alternatively define a single asexual
generation as the time between initiation of endospore
growth and spore release (fig. 4). In culture, endospore
growth involved five to nine rounds of mitosis, resulting
in sporangia with between 32 and 512 uninucleate endo-
spores. In nature, the number of mitotic divisions before
endospore release remains unknown, but assuming six
MBE
rounds of mitosis per sporangium, the ratio of frequency
of meiotic divisions to asexual endospore-to-endospore
generations, would be six times the meiosis:mitosis ratio.
For example, in figure 6, with q 5 1.0, the lowest estimate
of meiosis:mitosis was 1:154, but the ratio of meiosis:asex-
ual endospore-to-endospore generations would be 1:25.7.
This last ratio would imply that P. tapetis undergoes at least
25 rounds of sporangium formation for each round of mei-
osis. The change in the units of measure for an asexual
generation would, of course, have no effect on the time
between sexual generations or the probability of infection
of a host by an asexual propagule (fig. 6).
Regardless of the margins of error, P. tapetis is most likely
facultatively sexual and can probably undergo unlimited
rounds of asexual reproduction, much like a yeast. Out-
crossing rates in yeasts have been estimated at once per
3,000–9,000 asexual cycles for S. paradoxus (Tsai et al.
2008) and once per 50,000 asexual cycles for Saccharomyces
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cerevisiae (Ruderfer et al. 2006). Some protists have an ob-
ligate sexual stage because of constraints such as morphol-
ogy (e.g., diatom frustule size), development (alternation of
generations in foraminiferans), or host relationships (e.g.,
some apicomplexans). Even given our wide range of param-
eters, if P. tapetis had a similar obligate sexual cycle, we
would have expected to see a lower bound for the number
of asexual cycles per sexual cycle.
Host Relationship and Demographics
Having isolated P. tapetis 126 times from bivalve guts, we
suspect that guts are its main habitat. Anurofeca richardsi,
the closest relative to P. tapetis with any in situ observa-
tions, grows and divides in tadpole digestive tracts (Beebee
and Wong 1993). P. tapetis is unlikely to be as abundant in
the water column as it is within the digestive tract because,
in repeated attempts, we never isolated it from the undi-
luted mantle cavity fluid of bivalves even when it could be
cultured from the guts of the same animal. It is not known
how long P. tapetis resides in its host’s digestive tract, nor
has its growth there been physically observed. That P. ta-
petis may have just been ingested by bivalves as food is pos-
sible but food can pass through a bivalve digestive tract
within a day (Ren et al. 2006), and the high frequency
of isolation of P. tapetis would then have to be explained
by a high incidence in the water column. To reproduce suc-
cessfully in the gut P. tapetis would need a mechanism for
retention beyond the day needed for passage of food be-
cause, based on its growth in culture, P. tapetis endospores
took 3–5 days to grow to the reproductive stage. Con-
versely, P. tapetis’s variation in abundance among even co-
habiting hosts suggests that it comes and goes rather than
accumulating continuously over the lifespan of the host
(table 1).
According to our estimates of the frequency of sex, in-
fection of a host animal does not depend on a sexual stage.
If P. tapetis undergoes about two mitotic divisions per day
in the host as it does in culture, it can undergo approxi-
mately 700 mitotic divisions per year. Assuming an average
retention time of a year and our median estimates of
2023
Marshall and Berbee · doi:10.1093/molbev/msq078
frequency of sex, P. tapetis must pass through several hosts
before mating. If sex was obligate and infection limited to
sexually derived resting spores or zygotes, the retention
time necessary before sexual reproduction could equal
or surpass the host lifespan (fig. 6).
The average level of silent-site pairwise nucleotide diver-
sity (hw) was within the range but lower than the average
value of 0.057 of other unicellular eukaryotes reported by
Lynch (2006). The effective population size of P. tapetis was
on the order of 106 to 107, which is typical for unicellular
eukaryotes (Lynch 2006). Effective population sizes are
based on numbers of individuals expected to contribute
progeny to the next generation. Because many cells die
and do not contribute to the gene pool, the effective size
is lower than the censused population. Pseudoperkinsus ta-
petis can grow and divide for a few days in unsupplemented
seawater but cells arrest at uni- and binucleate stages.
Reinfection is probably a limiting factor in P. tapetis’s de-
mographics. The envisioned challenges of colonization and
reinfection faced by symbionts such as P. tapetis may be
offset by factors, such as reduced competition, ample sup-
ply of nutrients, or the ability to proliferate and mate once
past the reinfection hurdle.
Our finding of a large population with evidence of re-
combination suggests that P. tapetis is a mostly overlooked
endemic of a wide host range of shellfish and other inver-
tebrates rather than a recent colonizer. Nucleotide diversity
and effective population size decrease during population
bottlenecks such as recent colonization (Smith 1991; Rich
and Ayala 2000). The presence of a recombining population
also suggests a natural ecological niche (Campbell et al.
2005). For example, recombination in Giardia was shown
once isolates were collected from an endemic area with
high transmission rates and mixed infections (Cooper
et al. 2007). Although most frequently isolated from bi-
valves (Figueras et al. 2000; Takishita et al. 2008), Pseudo-
perkinsus showed no obvious detriment to its hosts. We
found no evidence of host specificity based on the distri-
bution of haplotypes.
Conclusions
We have demonstrated for the first time among any mem-
ber of the basal unicellular opisthokont lineages that sexual
recombination occurs and that sex is most likely faculta-
tive. According to our estimates of a low frequency of
sex, which were based on a small amount of data, P. tapetis
most likely enters the host as an asexually derived endo-
spore. Ichthyosporeans are one of few host-dependent
lineages among the opisthokonts. Phylogenetically, multi-
cellular animals originated after the ichthyosporean lineage
diverged and so the earliest ichthyosporeans could not
have had metazoan hosts. The lack of host specificity in
P. tapetis may reflect the relatively recent evolution of ich-
thyosporean dependence on metazoan hosts. Our data are
the first to reveal ploidy in any member of the unicellular
relatives of animals. A potentially haploid lineage at the
base of the metazoan tree contrasts with the diploid ani-
2024
MBE
mals, and if other unicellular opisthokonts are haploid as
well, then haploid dominant life cycles may have been the
ancestral condition of the metazoan stem lineage.
Supplementary Material
Supplementary tables S1 and S2 are available at Molecular Bi-
ology and Evolution online (http://www.mbe.oxfordjournals
.org/).
Acknowledgments
We thank S. Otto for helpful discussions on coalescent the-
ory, W. Maddison for Poisson probability calculations, and
L. Rieseberg and several anonymous reviewers for com-
ments on earlier versions of this manuscript. This work
was supported by the Natural Sciences and Research Coun-
cil of Canada and the Bamfield Marine Sciences Centre.
Downloaded from http://mbe.oxfordjournals.org/ by guest on September 15, 2012
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