ROTIFERA XIV
Fifteen species in one: deciphering the Brachionus plicatilis
species complex (Rotifera, Monogononta) through DNA
taxonomy
Scott Mills
.
J. Arturo Alca
´
ntara-Rodrı
´
guez
.
Jorge Ciros-Pe
´
rez
.
Africa Go
´
mez
.
Atsushi Hagiwara
.
Kayla Hinson Galindo
.
Christian D. Jersabek
.
Reza Malekzadeh-Viayeh
.
Francesca Leasi
.
Jae-Seong Lee
.
David B. Mark Welch
.
Spiros Papakostas
.
Simone Riss
.
Hendrik Segers
.
Manuel Serra
.
Russell Shiel
.
Radoslav Smolak
.
Terry W. Snell
.
Claus-Peter Stelzer
.
Cuong Q. Tang
.
Robert L. Wallace
.
Diego Fontaneto
.
Elizabeth J. Walsh
Received: 7 December 2015 / Revised: 15 February 2016 / Accepted: 28 February 2016 / Published online: 5 April 2016
Ó Springer International Publishing Switzerland 2016
Abstract Understanding patterns and processes in
biological diversity is a critical task given current and
rapid environmental change. Such knowledge is even
more essential when the taxa under consideration are
important ecological and evolutionary models. One of
these cases is the monogonont rotifer cryptic species
complex Brachionus plicatilis, which is by far the
most extensively studied group of rotifers, is widely
used in aquaculture, and is known to host a large
amount of unresolved diversity. Here we collate a
dataset of previously available and newly generated
sequences of COI and ITS1 for 1273 isolates of the B.
plicatilis complex and apply three approaches in DNA
taxonomy (i.e. ABGD, PTP, and GMYC) to identify
and provide support for the existence of 15 species
within the complex. We used these results to explore
phylogenetic signal in morphometric and ecological
traits, and to understand correlation among the traits
using phylogenetic comparative models. Our results
support niche conservatism for some traits (e.g. body
length) and phylogenetic plasticity for others (e.g.
genome size).
Guest editors: M. Devetter, D. Fontaneto, C. D. Jersabek,
D. B. Mark Welch, L. May & E. J. Walsh / Evolving rotifers,
evolving science
Diego Fontaneto and Elizabeth J. Walsh have contributed
equally to this work.
Electronic supplementary material The online version of
this article (doi:10.1007/s10750-016-2725-7) contains supple-
mentary material, which is available to authorized users.
S. Mills
James Cook University, 1 James Cook Drive,
Townsville 4811, Australia
J. Arturo Alca
´
ntara-Rodrı
´
guez J. Ciros-Pe
´
rez
Proyecto de Investigacio
´
n en Limnologı
´
a Tropical, FES
Iztacala, Universidad Nacional Auto
´
noma de Me
´
xico,
Mexico City, Mexico
A. Go
´
mez
School of Biological, Biomedical and Environmental
Sciences, University of Hull, Hull HU6 7RX, UK
A. Hagiwara
Graduate School of Fisheries and Environmental
Sciences, Nagasaki University, Nagasaki 852-8521, Japan
K. H. Galindo E. J. Walsh
Department of Biological Sciences, University of Texas at
El Paso, El Paso, USA
C. D. Jersabek
Department of Organismal Biology, University of
Salzburg, 5020 Salzburg, Austria
123
Hydrobiologia (2017) 796:39–58
DOI 10.1007/s10750-016-2725-7
Keywords Biodiversity COI Cryptic species
Evolution ITS1 Phylogenetic comparative
methods Zooplankton
Introduction
The occurrence of complexes of cryptic species—
groups of species that are not confidently distinguish-
able based only on morphology—has become widely
recognised in biodiversity analyses (Knowlton, 1993;
Bickford et al., 2007). The revolution brought by
efficient DNA sequencing technologies has driven an
explosion of studies on biodiversity, unmasking
hidden morphological diversity and revealing that
cryptic species are common and widespread across all
animal phyla (Pfenninger & Schwenk, 2007; Trontelj
& Fiser, 2009). While deciphering hidden diversity in
species complexes remains a taxonomic challenge, it
is crucial to address important questions in speciation
research to understand patterns and processes in
biodiversity (Butlin et al., 2009).
Phylum Rotifera is one of several phyla with a high
level of cryptic diversity (Fontaneto et al., 2009;
Garcı
´
a-Morales & Elı
´
as-Gutie
´
rrez, 2013; Gabaldo
´
n
et al., 2016). Cryptic diversity is expected in rotifers,
due to the small size of these animals, the paucity of
taxonomically relevant morphological features, and
the scarcity of rotifer taxonomists (Wallace et al.,
2006). Moreover, the reliance of rotifers on chemical
communication in species recognition (Snell, 1998)
may contribute to the prevalence of morphological
cryptic diversity. One clear example of cryptic diver-
sity in the phylum is the species complex Brachionus
plicatilis Mu
¨
ller, 1786, a cosmopolitan taxon with an
affinity for saline environments. Here we report an
extensive study undertaken to unravel the hidden
diversity within this species complex.
Two morphotypes of B. plicatilis were reported as
early as the 19th century when Ehrenberg ascribed the
name Brachionus muelleri Ehrenberg, 1834, as dis-
tinct from the first record for the species complex,
B. plicatilis (although the former name is now
considered a junior synonym of the latter). A modern
discussion of diversity in B. plicatilis began when two
strains with differing morphological and ecological
characteristics were recognised as the L (large) and S
(small) types (Oogami, 1976). From the early 1980s, it
became increasingly clear that the morphological and
genetic differences between the L and S strains
supported the hypothesis that the two morphotypes
should be recognised as separate species. Serra &
Miracle (1983) noted marked seasonal cyclomorphosis
in individuals from Spanish water bodies commenting
R. Malekzadeh-Viayeh
Artemia and Aquatic Research Institute, Urmia
University, Urmia, Iran
F. Leasi
Department of Invertebrate Zoology, Smithsonian
National Museum of Natural History, Washington, DC,
USA
J.-S. Lee
Department of Biological Science, College of Science,
Sungkyunkwan University, Suwon 16419, South Korea
D. B. Mark Welch
Josephine Bay Paul Center for Comparative Molecular
Biology and Evolution, Marine Biological Laboratory,
Woods Hole, MA, USA
S. Papakostas
Division of Genetics and Physiology, Department of
Biology, University of Turku, Turku, Finland
S. Riss C.-P. Stelzer
Research Institute for Limnology, University of
Innsbruck, 5310 Mondsee, Austria
H. Segers
OD Nature, Royal Belgian Institute of Natural Sciences,
1000 Brussels, Belgium
M. Serra
Institut Cavanilles de Biodiversitat i Biologia Evolutiva,
Universitat de Vale
`
ncia, 46071 Valencia, Spain
R. Shiel
Biological Sciences, University of Adelaide, Adelaide,
SA 5005, Australia
R. Smolak
Department of Ecology, Faculty of Humanities and
Natural Sciences, Presov University, 081 16 Presov,
Slovakia
T. W. Snell
School of Biology, Georgia Institute of Technology,
Atlanta, GA 30332-0230, USA
C. Q. Tang
Department of Life Sciences, The Natural History
Museum, Cromwell Road, London SW7 5BD, UK
40 Hydrobiologia (2017) 796:39–58
123
that, while B. plicatilis populations were thought to
exhibit high levels of phenotypic plasticity in their
natural habitat, laboratory clones founded from single
individuals could be readily distinguished biometri-
cally. They also noted a good correlation between
biometric classification and spatial distribution of wild
populations, hypothesising that some of their clones
may constitute a ‘‘well-differentiated genetic race’’.
The idea of discriminatory genetic structure within
what was considered a single species was further
supported by Snell & Carrillo (1984) who examined
13 strains of B. plicatilis sourced globally, concluding
that strain identity was the most important determin-
istic factor of size. Serra & Miracle (1987) supported
these observations, reporting that size in B. plicatilis
populations seemed to be largely under genetic
control. Furthermore, these authors noted that size
could be defined to a narrow range of biometric
deviations at different salinities and temperatures. In
the same year, King & Zhao (1987) reported a
substantial amount of genetic variation in three
enzyme loci between clones established from individ-
uals collected at different times from Soda Lake,
Nevada (USA). Other phenotypic traits provided
evidence for distinct species. For example, some
members of the species complex retain their resting
eggs within the body, while others employ a thin
thread to hold them outside their body (Serrano et al.,
1989).
The existence of cryptic species within B. plicatilis
was reinforced by Fu et al. (1991a), who examined 67
isolates from around the globe and showed that they
could be clearly classified into large (L) and small
(S) morphotypes based upon morphometric analysis
alone. In a second study, the same group clearly
discriminated between L and S strains on a genetic
basis and concluded that at least two species existed
(Fu et al., 1991b). Additional evidence for the
existence of at least two species within the taxon
came from the examination of chromosomes: L and S
morphotypes have karyotypes of 2n = 22 and
2n = 25, respectively (Rumengan et al., 1991,
1993). The size discontinuities between L and S
morphotypes were shown to correspond to beha-
vioural reproductive isolation between these groups
(Snell & Hawkinson, 1983). Snell (1989) showed how
male mate recognition could be used as a means of
establishing species boundaries in monogonont roti-
fers in this case. Both Fu et al. (1993) and Go
´
mez &
Serra (1995) also identified reproductive isolation
between the L and S types based on male mating
behaviour. Thus, in reviewing morphological, beha-
vioural, and genetic studies, Segers (1995) concluded
that the L and S strains could be defined as two distinct
species, namely B. plicatilis sensu stricto (s.s.) and
Brachionus rotundiformis Tschugunoff, 1921,
respectively.
Further investigations by Go
´
mez & Serra (1995),
Go
´
mez et al. (1995), Go
´
mez & Snell (1996), Serra
et al. (1998), and Ortells et al. (2000) using molecular
markers and reproductive isolation tests revealed that
several cryptic species could be ascribed to both
B. plicatilis and B. rotundiformis . This revelation
culminated in a paper by Ciros-Pe
´
rez et al. (2001a)
that used morphological, ecological, and genetic
differences to support B. plicatilis s.s. and B. rotundi-
formis and to introduce a medium size type, desig-
nated SM, to the species complex with the description
of Brachionus ibericus Ciros-Pe
´
rez, Go
´
mez & Serra,
2001. At this stage, three groups were known: L with
B. plicatilis s.s., SM with B. ibericus, and SS (here so
called with two capital ‘s’ to be clearly differentiated
from the S strains) with B. rotundiformis (Fig. 1).
A phylogenetic analysis of mitochondrial and
nuclear gene sequences (namely COI and ITS1) on a
worldwide dataset supported an ancient differentiation
of this rotifer lineage into at least nine species, often
sympatric, which were clustered into the morpholog-
ically recognised L, SM, and SS morphotypes (Go
´
mez
et al., 2002). Suatoni et al. (2006) suggested the
existence of 14–16 species across the three clades,
based on DNA sequence data and the high degree of
concordance between genealogical and reproductive
isolation (based on experimental trials). Supporting
this diversity, genetic and phenotypic data were then
used to describe two additional species: Brachionus
manjavacas Fontaneto, Giordani, Melone & Serra,
2007, within the L type (Fontaneto et al., 2007) and
Brachionus koreanus Hwang, Dahms, Park & Lee,
2013 within the SM type (Hwang et al., 2013). Finally,
another species, already described as Brachionus
R. L. Wallace
Department of Biology, Ripon College, Ripon, USA
D. Fontaneto (&)
Institute of Ecosystem Study, National Research Council
of Italy, 28922 Verbania Pallanza, Italy
e-mail: d.fontaneto@ise.cnr.it
Hydrobiologia (2017) 796:39–58 41
123
Fig. 1 Photomicrographs
of three representative
lineages of the Brachionus
plicatilis species complex.
A, B, C) dorsal view; D, E,
F) lateral view; G, H, I)
ventral view. A, D, G Large
strain, clone BUSCL (clade
L1 in Figs. 2–5); B, E,
H Medium strain, clone
MULCL (clade SM4); C, F,
I) Small strain, clone
TOWCL (clade SS1). Scale
bar = 100 lm
42 Hydrobiologia (2017) 796:39–58
123
asplanchnoidis Charin, 1947, were known to be a
member of the group (Kutikova, 1970; Segers, 1995;
Jersabek & Bolortsetseg, 2010); however, no DNA
sequences could be unambiguously attributed to it.
Thus, a sizable amount of analyses using molecular,
morphological, ecological, and reproductive isolation
suggests that there are many putative species within
the B. plicatilis complex. However, only six species
have been formally described (in chronological order):
B. plicatilis s.s., B. rotundiformi s, B. asplanchnoidis,
B. ibericus, B. manjavacas, and B. koreanus, respec-
tively, by Mu
¨
ller (1786), Tschungunoff (1921), Charin
(1947), Ciros-Pe
´
rez et al. (2001a), Fontaneto et al.
(2007), and Hwang et al. (2013). Nevertheless, there
are additional clades that may correspond to putative
new species and that have been designated by the
scientific community simply as ‘‘Brachionus sp.
‘Locality’’’, where ‘Locality’ refers to the place where
the samples were first collected. Examples of this
designation include Brachionus sp. ‘Almenara’
(Ortells et al., 2000;Go
´
mez et al., 2002), Brachionus
sp. ‘Nevada’ (Go
´
mez et al., 2002), and Brachionus sp.
‘Mexico’ (Alca
´
ntara-Rodrı
´
guez et al., 2012).
In an effort to clarify the systematics of the
B. plicatilis species complex, we present an analysis
of the most extensive dataset on genetic diversity in
the species complex. The first aim of our contribution
is to provide a clear phylogenetic structure to support
identification and designation of species in the com-
plex through the use of several approaches in DNA
taxonomy. Our second aim is to present a study of the
evolutionary relationships among the species in the
complex for a comparative analysis exploring the
phylogenetic signal of biological traits and correla-
tions among species-specific traits of the different
species. The B. plicatilis species complex is by far the
most extensively studied group of rotifers, and these
animals have been used to investigate a wide variety of
phenomena including ecological interactions (Ciros-
Pe
´
rez et al., 2001b, 2004, 2015; Montero-Pau et al.,
2011; Gabaldon et al., 2015), toxicology (Serrano
et al., 1986; Snell & Persoone, 1989; Dahms et al.,
2011), osmoregulation (Lowe et al., 2005), local
adaptation (Campillo et al., 2009; Alca
´
ntara-Rodrı
´
-
guez et al., 2012), the evolution of sex (Carmona et al.,
2009), phylogeography (Go
´
mez et al., 2000, 2007;
Mills et al., 2007), ageing (Snell et al., 2015), and
evolutionary processes (Stelzer et al., 2011; Fontaneto
et al., 2012; Tang et al., 2014a). In addition, due to the
ease and low cost of producing highly dense cultures
of these rotifers, members of this species complex
have been widely used in aquaculture as a source of
live feed for larval crustaceans and fishes (Fukusho,
1983; Watanabe et al., 1983; Lubzens & Zmora,
2003). We make use of this information to provide a
first assessment of the evolutionary trajectories of
biological and ecological traits in the B . plicatilis
species complex.
Methods
Data collection
We gathered all the DNA sequences for COI (Cy-
tochrome Oxidase c subunit I) and ITS1 (Internal
Transcribed Spacer 1) from members of the B. pli-
catilis species complex that were available in
GenBank in March 2015. To ensure the quality of
the data, we removed short sequences (4 sequences
shorter than 300 bp were removed from the COI
dataset), confirmed that the COI sequences lacked
internal stop codons (given that NCBI did not do it
automatically for the older sequences), investi-
gated that the maximum uncorrected genetic differ-
ence among the sequences was less than 40%, and
verified that the best BLAST hit for each sequence
was from a rotifer of the genus Brachionus.This
resulted in the retention of 811 COI and 184 ITS1
sequences. In addition, we sequenced COI and ITS1
from a total of 449 wild—caught individuals or
existing lab strains, using DNA extraction and gene
amplification protocols established for the species
complex more than a decade ago (Go
´
mez et al., 2002).
The full list of 1273 isolates used for the study and the
GenBank accession numbers of their COI and ITS1
sequences are provided in Supplementary File S1. All
newly obtained sequences were deposited in GenBank
with accession numbers from KU299052 to
KU299752. We did not include sequences from clades
15 and 16 of Suatoni et al. (2006), as they seem to be
outside the species complex, they have never been
found again, no voucher or lab cultures exist, and no
additional information is available for them.
In addition to DNA sequence data, we collected
contextual data for all 1273 isolates, when available.
These data included the name of the water body where
they were found, the country and continent of
Hydrobiologia (2017) 796:39–58 43
123
