ArticlePDF Available

Systematic study of Simocephalus s. str. species group (Cladocera: Daphniidae) from Taiwan by morphometric and molecular analyses.

Authors:

Abstract and Figures

There is some controversy regarding the traditional taxonomy of the Simocephalus sensu stricto species group. We conducted molecular and morphometric analyses to differentiate the 3 species from this group found in Taiwan: S. vetulus (O.F. Müller, 1776), S. vetuloides Sars, 1898, and S. mixtus Sars, 1903. The landmark method was employed, followed by a transfer into 24 characteristic values for a principal component analysis (PCA), the results of which indicated morphometric overlap among these species. The dorsal angle, brood size, and body length were smallest in S. vetulus, medium in S. vetuloides, and largest in S. mixtus. In the Simocephalus sensu stricto group from Taiwan, the dorsal angle and body length were significantly correlated with brood size in a quadratic manner. In the molecular analysis, 98 specimens of Simocephalus were used, and the 641-bp mitochondrial DNA cytochrome oxidase subunit 1 sequence was employed as a marker to analyze the genetics of S. vetulus, S. vetuloides, S. mixtus, S. serrulatus (Koch, 1841), and S. heilongjiangensis Shi and Shi, 1994. Simocephalus vetulus, S. vetuloides, and S. mixtus shared several haplotypes, and the interspecific genetic distance was merely 0.00671-0.00785, which is within the range of intraspecific differences. We concluded that S. vetulus, S. vetuloides, and S. mixtus in Taiwan belong to the same species and should be treated as S. cf. vetulus. The number of species of Simocephalus in Taiwan is thus reduced to 3: S. cf. vetulus, S. serrulatus, and S. heilongjiangensis.
Content may be subject to copyright.
Systematic Study of the Simocephalus Sensu Stricto Species Group
(Cladocera: Daphniidae) from Taiwan by Morphometric and Molecular
Analyses
Shuh-Sen Young1,*, Mei-Hui Ni2, and Min-Yun Liu3
1Department of Applied Science, National Hsinchu University of Education, Hsinchu 300, Taiwan
2Hsinchu Municipal Hsinchu Elementary School, Hsinchu 300, Taiwan
3National Applied Research Laboratories, Taiwan Ocean Research Institute, Taipei 106, Taiwan
(Accepted September 9, 2011)
Shuh-Sen Young, Mei-Hui Ni, and Ming-Yun Liu (2012) Systematic study of the Simocephalus sensu stricto
species group (Cladocera: Daphniidae) from Taiwan by morphometric and molecular analyses. Zoological
Studies 51(2): 222-231. There is some controversy regarding the traditional taxonomy of the Simocephalus
sensu stricto species group. We conducted molecular and morphometric analyses to differentiate the 3 species
from this group found in Taiwan: S. vetulus (O.F. Müller, 1776), S. vetuloides Sars, 1898, and S. mixtus Sars,
1903. The landmark method was employed, followed by a transfer into 24 characteristic values for a principal
component analysis (PCA), the results of which indicated morphometric overlap among these species. The
dorsal angle, brood size, and body length were smallest in S. vetulus, medium in S. vetuloides, and largest
in S. mixtus. In the Simocephalus sensu stricto group from Taiwan, the dorsal angle and body length were
significantly correlated with brood size in a quadratic manner. In the molecular analysis, 98 specimens of
Simocephalus were used, and the 641-bp mitochondrial DNA cytochrome oxidase subunit 1 sequence was
employed as a marker to analyze the genetics of S. vetulus, S. vetuloides, S. mixtus, S. serrulatus (Koch, 1841),
and S. heilongjiangensis Shi and Shi, 1994. Simocephalus vetulus, S. vetuloides, and S. mixtus shared several
haplotypes, and the interspecic genetic distance was merely 0.00671-0.00785, which is within the range of
intraspecic differences. We concluded that S. vetulus, S. vetuloides, and S. mixtus in Taiwan belong to the
same species and should be treated as S. cf. vetulus. The number of species of Simocephalus in Taiwan is thus
reduced to 3: S. cf. vetulus, S. serrulatus, and S. heilongjiangensis.
http://zoolstud.sinica.edu.tw/Journals/51.2/222.pdf
Key words: Systematics, Biodiversity, Simocephalus, Freshwater zooplankton.
*To whom correspondence and reprint requests should be addressed. E-mail:shuh@mail.nhcue.edu.tw
The general morphologies of Simocephalus
vetulus (O.F. Müller, 1776), S. vetuloides Sars
1898, and S. mixtus Sars 1903 are very similar.
Sars (1916) first discriminated S. vetulus and
S. vetuloides based on the dorsoposterior valve
angle. After that, many authors defined S.
vetuloides by a more-protruding dorsal valve
margin and more-numerous and larger denticles
on the posterior dorsal valve margin compared
to S. vetulus (Uéno 1966, Chiang and Du 1979,
Yoon and Kim 1987 2000, Shi and Shi 1996, Kim
1998, Orlova-Bienkowskaja 2001, Tuo 2002).
Other authors treated S. vetuloides as a local form
(Johnson 1953) or as a synonym of S. vetulus
(Fryer 1957, Harding 1961, Sharma 1978, Negrea
1983, Michael and Sharma 1988). Sars (1903)
described S. mixtus as having a more-protruding (to
the rear) dorsal valve margin and larger denticles
on the posterior dorsal valve margin compared to
S. vetulus and S. vetuloides. Flössner (1972) and
Negrea (1983) treated S. mixtus as a synonym of
S. vetulus. After that, Orlova-Bienkowskaja (1998)
Zoological Studies 51(2): 222-231 (2012)
222
made a more-detailed revision and treated S.
mixtus as a valid species.
Orlova-Bienkowskaja (2001) proposed a
different method of discriminating S. vetulus, S.
vetuloides, and S. mixtus. She drew an inner circle
along the shell posterior, the diameter of which
and the prominence of the dorsal valve being key
features for identification. The shell posterior of
S. vetulus ends without an extending shell spine,
the inner circle is larger than in S. vetuloides and
S. mixtus, while S. mixtus has more-protruding
dorsal valves than S. vetuloides. The diameter
of the inner circle of S. mixtus is larger than the
prominence portion, and S. vetuloides differs from
S. mixtus in that the diameter of the inner circle of S.
vetuloides is smaller than the prominence portion.
In the past, many authors proposed S. vetulus
to be a cosmopolitan species rst des-cribed from
the Old World, as it was found in many areas,
with the exception of New Zealand and Australia
(Werestschagin 1923, Uéno 1927, Rylov 1930,
Hemsen 1952, Harding 1961, Manuilova 1964,
Uéno 1966, Chiang and Du 1979, Rajapaksa and
Fernando 1982, Boonsom 1984, Yoon and Kim
1987, Kim 1998, Mizuno and Takahashi 1991, Du
1993, Hann 1995, Shi and Shi 1996, Michael and
Sharm 1998, Tuo 2002). Orlova-Bienkowskaja
(2001) indicated that the distribution of S. vetulus
was limited to northern Africa and Europe,
while S. vetuloides had a limited distribution in
eastern Siberia. Outside of Africa, Europe, and
eastern Siberia, Simocephalus sensu stricto
comprises S. punctatus Orlova-Bienkowskaja,
1998, S. elizabethae (King, 1853), and S.
mixtus. Simocephalus mixtus is a cosmopolitan
species distributed in Asia, Eastern Europe,
North Africa, and North America. Simocephalus
(Coronocephalus) serrulatus (Koch, 1841) is
also regarded as a cosmopolitan species, as
it is distributed in Asia, Europe, Africa, North
America, South America, and Australia (Orlova-
Bienkowskaja 2001).
Based on the description by Orlova-
Bienkowskaja (2001) and other morphological
comparisons, Tuo (2002) described 3 species
of Simocephalus from Taiwan, S. serrulatus,
S. vetulus, and S. vetuloides. Since then, this
extensive collection has increased to include S.
heilongjiangensis Shi and Shi, 1994 and S. mixtus
Sars from southern Taiwan (Ni 2005). At some
collection sites, S. vetulus and S. vetuloides were
found simultaneously as were S. vetuloides and
S. mixtus (Ni 2005). Morphological similarities
among S. vetulus, S. vetuloides, and S. mixtus are
large, with the exception of the shape of the dorsal
valve. However, the shape of the dorsal valve
of cladocerans may be affected by the brooding
status, with growing embryos pushing the valve
more prominently outwards, than in individuals
without eggs.
The species level is recognized as the
basic unit of biodiversity (Mayer and Ashlock
1991). Nowadays, alpha taxonomy is still based
mainly on morphology. Morphometry is one of
several possible methods to determine species
and analyze morphological differences between
closely related species and populations (Chen et
al. 2010). With the advent of molecular technology
for DNA sequencing, morphologically cryptic
species have been increasingly revealed, and the
use of DNA markers as a new tool to overcome
morphological impediments was suggested (Tautz
et al. 2003). The ideal DNA-based identification
system (DNA barcodes) would employ a single
gene, and be suitable for any organism in the
taxonomic hierarchy. Folmer et al. (1994) de-
signed a universal primer for the mitochondrial
cytochrome oxidase subunit I (COI) gene, which
subsequently became a popular marker to study
invertebrates. Hebert et al. (2003), Tautz et al.
(2003), Blaxter (2004), Lefébure et al. (2006),
and Costa et al. (2007) suggested that the COI
gene appears to be an appropriate molecular
marker (as a DNA barcode) on several taxonomic
scales, but particularly at the species level. We
attempted to clarify the taxonomic status of S.
vetulus, S. vetuloides, and S. mixtus in Taiwan
by morphometric comparisons and used the
mitochondrial (mt)DNA COI gene marker as a new
character.
This paper is our 1st step dealing with
vetulus-like populations of Simocephalus in Taiwan,
which are currently regarded as conspecific to
the Palaearctic cosmopolitan species. We thus
attempted to improve the taxonomy of the genus
Simocephalus by solving a small piece of the
puzzle from the overall picture.
MATERIALS AND METHODS
Samples were taken from many temporary
freshwater bodies throughout Taiwan using a
plankton net. Each sample was fixed in 70%
ethanol (EtOH), later preserved in 95% EtOH and
stored at a low temperature (< -20°C). Within 72 h,
each raw sample was sorted and identied under
a stereomicroscope. In total, 187 individuals (170
Young et al. – Study of Simocephalus from Taiwan 223
(A)
(D)
(B)
(E)
(C)
with eggs) were collected in 2003 and 2004 and
used for the morphometric analysis: 45 individuals
of S. vetulus from 8 sites, 72 individuals of S.
vetuloides from 11 sites, and 70 individuals of
S. mixtus from 10 sites. From this set of 187
individuals, 72 individuals, including 22 individuals
of S. vetulus from 8 sites, 28 individuals of S.
vetuloides from 11 sites, and 22 individuals of S.
mixtus from 10 sites, were selected for the DNA
analysis. Additionally, 7 individuals of S. serrulatus
(Fig. 1) from 3 sites and 19 individuals of S.
heilongjiangensis (Fig. 1) from 5 sites were also
included in the DNA analysis. Daphnia similoides
Hudec, 1991 (Daphniidae) and Diaphanosoma
dubium Manuilova, 1964 (Sididae) from Taiwan
were analyzed in order to obtain outgroup
sequences.
Morphometric analysis
Lateral-view images of S. vetulus, S.
vetuloides, and S. mixtus were taken using a
digital camera under a stereomicroscope for the
morphometric study. Morphometric characters
were extracted from the photographic images, and
8 morphometric data points were used to construct
Fig. 1. General morphology of female Simocephalus with summer eggs found in Taiwan (all drawings are original). (A) S. vetulus; (B)
S. vetuloides; (C) S. mixtus; (D) S. serrulatus; (E) S. heilongjiangensis. The valve shape is the major difference among S. vetulus, S.
vetuloides, and S. mixtus; S. serrulatus has teeth on the top of its head, and S. heilongjiangensis has a different posterior end of the
valve. Scale bars = 0.1 mm.
224 Zoological Studies 51(2): 222-231 (2012)
24 length measurements, each of which was
divided by body length to obtain size-free ratios
(Fig. 2). The body length and dorsal valve angle
(Fig. 2) were also measured on the photographic
images, and the clutch size of each individual
was assessed under a microscope. SPSS vers.
10.0.1 (Chicago, IL, USA) was used to analyze
the numerical data. The data matrix was tested
using the Kaiser-Meyer-Olkin (KMO) measure
of sampling adequacy and by the Bartlett X2 test
prior to the principle component analysis (PCA).
For individuals with eggs, Pearson’s correlation
analyses and non-linear regressions among the
dorsal angle, body length, and clutch size were
carried out.
DNA extraction, amplication, and sequencing
Total genomic DNA was extracted using
Chelex (InstaGene Matrix BIO-RAD 7326030,
Bio-Rad Laboratories, Hercules USA) from single
animals. Each animal was taken from 95% EtOH
and placed into pure water for 1 h for cleaning.
After that, each animal was placed at the bottom
of a 0.5-ml centrifuge tube for 30 min to dry in a
speed vacuum-drying system. Dried samples
were then ground up by needles, and 50 μl of a 5%
Chelex solution was used to extract the DNA by
incubation at 56°C for 2-3 h, followed by incubation
at 90°C for 8 min. For each polymerase chain
reaction (PCR), 5 μl of upper cleaning was used as
the DNA template after centrifugation at 104 rpm
(9168g) for 3 min.
We employed the universal primers, LCO
1490 (5'-GGTCAACAAATCATAAAGATATTGG-3')
and HCO2918 (5'-TAAACTTCAGGGTGACCAA
AAAATCA-3') (Folmer et al. 1994), to amplify the
mitochondrial COI gene by a PCR. Each PCR
sample had a total volume of 50 μl and consisted
of pH 9.2 buffer solution (50 mM Tris-HCl, 16 mM
ammonium sulfate, 2.5 mM MgCl2, and 0.1%
Tween 20), 5 pM of each primer, 50 μM of dNTPs,
2 units of Taq DNA polymerase (super Therm
DNA polymerase, Bio-Taq, BioKit Biotechnology,
Miaoli Taiwan), and 10-50 ng of genomic DNA.
The PCRs were performed in an Eppendorf
Mastercycler gradient 384 machine (Eppendorf,
Hamburg, Germany). Thermocycling began with
5 min of preheating and continued with 35 cycles
at 94°C for 30 s, primer annealing at 51°C for
45 s, and extension at 72°C for 45 s; followed by
incubation at 72°C for 10 min for full extension
of the DNA and ended with 4°C holding. PCR
products were electrophoresed in 2% agarose
gels, after which the gels were stained with
ethidium bromide (EtBr) and photographed under
an ultraviolet light box. DNA fragments were
excised from the gel and extracted using a 1-4-
3 DNA extraction kit (Gene-Spin, Protech, Taipei,
Taiwan) to obtain purified DNA. Sequences of
DNA fragments were resolved on an ABI3730
automated sequencer (Applied Biosystems,
Carlsbad, California USA) using 20-50 ng of tem-
plate with 5 pM of the LCO1490 primer.
Alignment, genetic diversity, and phylogeny
After a search of GenBank, all COI sequences
of Simocephalus were downloaded and aligned
with our sequences. The download sequences
included S. vetulus from the UK (accession no.,
DQ889172: Costa et al. 2007), S. cf. punctatus
from Mexico and Guatemala (EU702310 and
EU702282, Elias-Gutierrez et al. 2008), S.
cf. exspinosus from Mexico and Guatemala
A
B
C
H
G
F
ED
60° 60°
Fig. 2. Morphometry of each specimen extracted from 8
data points (A-H), from which we constructed 24 length
measurements; each length measurement was then divided by
body length (AE) to obtain size-free ratios. The angle between
lines AE and ED was taken as the dorsal valve angle.
Young et al. – Study of Simocephalus from Taiwan 225
(EU702296 and EU702279, Elias-Gutierrez et al.
2008), S. cf. mixtus from Mexico and Guatemala
(EU702305 and EU702281, Elias-Gutierrez et
al. 2008), and S. serrulatus from Mexico and
Guatemala (EU702312, Elias-Gutierrez et al.
2008). COI gene sequences were aligned by eye
using the BioEdit program vers. 7.0.2 (Hall 1999).
We calculated the haplotype diversity (Hd, Nei
1987), nucleotide diversity (π, Nei 1987), genetic
distance (Dxy, Nei 1987), and average genetic
distances between each pair of species using
MEGA 3 vers. 3.0 (Kumar et al. 2004). Daphnia
similoides and Diaphanosoma dubium were used
as outgroups, and the phylogenetic tree was
derived using all sequences by the Neighbor-
joining (NJ) and maximum-parsimony (MP)
methods (Saitou and Nei 1987) based on Kimura
2-parameter (K2P) distances with 1000 bootstraps
using MEGA 3.
RESULTS
Morphometric comparisons of Simocephalus
vetulus, S. vetuloides, and S. mixtus
The KMO value for the morphometric data
matrix was 0.81, and Bartlett’s X2 was 2583.96
(d.f. = 276; p = 0.000), demonstrating the
suitability of the PCA. After the PCA, 91% of
the variance was explained by the 1st, 2nd, and
3rd components combined. On the 1st and 2nd
component plots, S. vetulus and S. mixtus were
separated from each other, but S. vetuloides was
mixed with both groups; thus, they did not separate
very well into 3 different species (Fig. 3).
Simocephalus vetulus individuals with eggs
(n = 170) (clutch sizes ranged 1-4, dorsal valve
angle ranged 39.5°-74.8°) had fewer eggs than the
2 other species; S. vetuloides (clutch sizes ranged
1-12, dorsal valve angle ranged 41.5°-69.5°) was
intermediate; and S. mixtus (clutch sizes ranged
1-30; dorsal valve angle ranged 63.4°-97.5°) had
the most eggs. In a pooled analysis of these
3 species, Pearson’s correlation between the
dorsal valve angle and clutch size was r = 0.725
(p = 0.000), and between body length and clutch
size was r = 0.70 (p = 0.000). The relationship
between clutch size (Y) and dorsal valve angle (X)
t a quadratic function Y = 0.0088X2 - 0.9091X +
25.3361 (r2 = 0.53), and the one between clutch
size (Y) and body length (X) also fit a quadratic
function Y = 9.81X2 - 20.48X + 12.00 (r2 = 0.49).
Hence, irrespective of the species, clutch size was
positively correlated with the dorsal valve angle
and body length. The valve shape was not a
species-specic character, but rather it depended
on the clutch size.
Molecular analysis of COI sequences
We used 110 COI sequences from S. vetulus
(n = 22), S. vetuloides (n = 28), S. mixtus (n = 10),
S. serrulatus (n = 7), S. heilongjiangensis (n = 19),
Daphnia similoides (n = 5), and Diaphanosoma
dubium (n = 7) for the phylogenetic analysis. Each
sequence was 641 bp long. Twelve haplotypes
were detected for the 5 species of Simocephalus
with 151 segregation sites; the genetic diversity,
Hd, was 0.891, and the nucleotide diversity, π, was
0.07049. Simocephalus vetulus had 4 haplotypes
from 8 sites (Hd = 0.576), S. vetuloides had 6
haplotypes from 11 sites (Hd = 0.802), S. mixtus
had 4 haplotypes from 9 sites (Hd = 0.636),
S. serrulatus had 2 haplotypes from 3 sites
(Hd = 0.571), and S. heilongjiangensis had 3
haplotypes from 6 sites (Hd = 0.374) (Table 1).
Genetic distances (Dxy) between each pair of
species based on the COI gene ranged 0.00671-
0.1604 (Table 2). Genetic distances among S.
vetulus, S. vetuloides, and S. mixtus were all
< 0.01, while those between S. serrulatus and the
other species were > 0.15, and those between
3
2
1
0
-1
-2
PCA 1
-4 -3
S. vetulus S. mixtus S. vetuloides
-2 -1 0
PCA 2
1234
Fig. 3. Results of the principal component analysis of the
morphometric dataset: 1st and 2nd principle component plot.
Simocephalus vetulus and S. mixtus were well separated with
a distribution gap, while S. vetuloides lled the gap and mixed
with those 2 species.
226 Zoological Studies 51(2): 222-231 (2012)
S. heilongjiangensis and the other species were
> 0.14.
In the phylogenetic NJ tree (Fig. 4), S.
vetulus, S. vetuloides, and S. mixtus (hap a-g)
were mixed together as a well-supported group
with a bootstrap value of 99%. Simocephalus
serrulatus (hap k-l) and S. heilongjiangensis (hap
h-j) were well separated, with each group being
supported by a 99% bootstrap value. The dorsal
valve shape variation was not associated with
genetic differences based on the COI gene. The
most protruding valve shape (S. mixtus) was
common in haplotypes a and b. Valve shapes of
S. vetulus and S. vetuloides were also common
Table 1. Haplotypes (Hap) of each species of Simocephalus and their collection sites
Haplotype nCollection sites
S. vetulus 22 8 collection sites; HD = 0.576; π = 0.00806
Hap a14 scA (3), scB (3), scC (2), zb (6)
Hap e2 dgA (2)
Hap f3 dy (3)
Hap g3 scE (2), hsB (1)
S. vetuloides 28 11 collection sites; HD = 0.802; π = 0.00777
Hap a10 hsA (3), scD (3), sf (1), xse (3)
Hap b5dd (1), khC (1), mn (3)
Hap c3 lj (3)
Hap d6 bs (6)
Hap e2 khB (2)
Hap g2 gA (2)
S. mixtus 22 10 collection sites; HD = 0.636; π = 0.00535
Hap a12 gA (2), dh (3), dy (3), hsA (1), scF (3)
Hap b6 dd (2), dy (1), tt (3)
Hap e3 dgB (3)
Hap g1 gs (1)
S. serrulatus 73 collection sites; HD = 0.571; π = 0.00357
Hap k4 mf (4)
Hap l3 gs (1), sf (2)
S. heilongjiangensis 19 6 collection sites; HD = 0.374; π = 0.00140
Hap h15 pjA (3), pjB (4), pjC (4), pjD (4)
Hap i2 khA (2)
Hap j2 khA (2)
bs: Baoshan (Hsinchu County); dd: Dadu (Taichung County); dgA-B: Dongang A-B (Pingtung County); dh: Dahu
(Miaoli County); dy: Dayuan (Taoyuan County); gA: Green Grass Lake (Hsinchu City); gs: Guanxi (Hsinchu
County); hsA-B: Hengshan A-B (Hsinchu County); khA-C: Kaohsiung City A-C; lj: Longjing (Taichung County);
mf: Minfu (Hsinchu city); mn: Meinong (Kaohsiung County); pjA-D: Pingzhen A-D (Taoyuan County); scA-E:
Hsinchu City A-E; sf: Shinfeng (Hsinchu County); tt: Taitung city; xse: Xiangshan (Hsinchu City); zb: Zhubei
(Hsinchu County).
Table 2. Genetic distances (Dxy) among Simocephalus species from Taiwan based on
mitochondrial DNA cytochrome oxidase subunit I sequences
S. vetuloides S. mixtus S. vetulus S. serrulatus
S. vetuloides ----
S. mixtus 0.00671
S. vetulus 0.00785 0.00698
S. serrulatus 0.15550 0.15572 0.15473
S. heilongjiangensis 0.16017 0.16046 0.15945 0.14391
Young et al. – Study of Simocephalus from Taiwan 227
in haplotype b. Haplotypes e and g were shared
by all 3 morphospecies (Fig. 5, Table 1). We
reconstructed the phylogenetic trees by including
both our sequences and downloaded sequences,
and obtained NJ and MP phylogenetic trees with
similar tree structures (Fig. 6). Haplotypes a-g
from Taiwan were all placed in the same group.
DISCUSSION
DNA barcoding can be helpful in species
identication within cryptic species groups (Hebert
et al. 2004, Belyaeva and Taylor 2009). In general,
sequence divergences are much lower among
individuals of a species than between closely
related species. For example, congeneric species
of moths exhibit an average sequence divergence
of 6.5% in the mitochondrial COI gene, whereas
divergences among conspecific individuals
average only 0.25% (Moore 1995, Hebert et al.
2004). Similar values were obtained in birds,
with intraspecific divergences of COI averaging
0.27%, whereas congener divergences averaged
7.93% (Hebert and Stoeckle et al. 2004). Among
1781 congeneric species pairs of crustaceans,
only 1.3% had COI gene divergences of < 2%,
13.4% had COI gene divergences ranging 4%-
8%, and 81.8% had COI gene divergences
ranging 8%-32% (Hebert et al. 2003). In a study
of the scale of intercontinental divergence for the
cladoceran genus Daphnia, Adamowicz et al.
(2009) observed a pairwise sequence divergence
within the D. obtusa complex of up to a maximum
of 16.9%, with divergences of up to 19% within
the D. longispina complex. In our study, S.
serrulatus and S. heilongjiangensis showed 14%-
16% COI divergence from each other and from
Simocephalus sensu stricto. These interspecific
differences were similar to most crustaceans
(Hebert et al. 2003).
Based on the morphological differences
described by Orlova-Bienkowskaja (2001), 3
species - S. vetulus, S. vetuloides, and S. mixtus
- were previously recorded in Taiwan. Indeed, our
morphometric analysis of the valve shape revealed
a significant difference between S. vetulus
and S. mixtus from Taiwan, which appeared
to support their taxonomic status as different
species. However, when all 3 putative species
were included in the analysis, the PCA did not
separate S. vetulus, S. vetuloides, and S. mixtus
from one another, as they formed a morphological
continuum. This is consistent with a single
morphologically variable species. Furthermore,
differences in valve shape among S. vetulus, S.
vetuloides, and S. mixtus collected in Taiwan were
not associated with genetic variations. The genetic
distances in COI among them were very small
(0.6%-0.8%), a divergence level that corresponds
Fig. 4. Phylogenetic tree for Simocephalus species in Taiwan, derived using the Neighbor-joining (NJ) method based on mitochondrial
(mt)DNA cytochrome oxidase subunit I (COI) sequences. The numbers indicate support values for 1000 bootstrap calculations.
0.02
86
57
69
99
99
71
99
99
79
99
Diaphanosoma dubium
Daphnia similoides
Daphnia similoides
Hap d: S. vetuloides, S. mixtus, S. vetulus
Hap e: S. vetuloides, S. mixtus, S. vetulus
Hap g: S. vetuloides
Hap f: S. vetulus
Hap a: S. vetuloides, S. mixtus, S. vetulus
Hap b: S. vetuloides, S. mixtus
Hap c: S. vetuloides
Hap k: S. serrulatus
Hap l: S. serrulatus
Hap h: S. heilongjiangensis
Hap i: S. heilongjiangensis
Hap j: S. heilongjiangensis
228 Zoological Studies 51(2): 222-231 (2012)
Fig. 6. Reconstructed phylogenetic trees of Simocephalus. Sequences from GenBank were included in this analysis: S. vetulus
(accession no., DQ889172) from the UK, S. cf. punctatus (EU702310 and EU702282) from Mexico and Guatemala, S. cf. exspinosus
(EU702296 and EU702279) from Mexico and Guatemala, S. cf. mixtus (EU702305 and EU702281) from Mexico and Guatemala, and
S. serrulatus (EU702312) from Mexico and Guatemala. Both the Neighbor-joining (NJ) and maximum-parsimony (MP) trees shared
similar branching structures. Haplotypes a-f from our study were all grouped together.
Fig. 5. Dorsal valve shapes of different haplotypes belonging to Simocephalus vetulus, S. vetuloides, and S. mixtus. Haplotypes a, b, e,
and g have different valve shapes with large-scale variations.
scC-1
scA-2
sf-1
dh-1
ha-5
Hap a
dgA-1
khB-4
dgB-1
dy-2
scE-3 gA-1
hs3-3
gs-1
Hap e
Hap d
bs-1
Hap f
Hap g
Hap b
Hap c
scD-1
scf-1
hs3-1
dd-3
khC-5 dd-1
tt-2
1j-2
mn-2
Hap d (*)
Hap g (*)
Hap e (*)
Hap f (*)
Hap a (*)
Hap b (*)
Hap c (*)
Hap d (*)
Hap g (*)
Hap e (*)
Hap f (*)
Hap a (*)
Hap b (*)
Hap c (*)
Simocephalus vetulus (+)
Simocephalus cf. punctatus (#)
Simocephalus punctatus (#)
Simocephalus cf. exspinosus (#)
Simocephalus cf. exspinosus (#)
Simocephalus cf. mixtus (#)
Simocephalus cf. mixtus (#)
Simocephalus cf. punctatus (#)
Simocephalus punctatus (#)
Simocephalus cf. exspinosus (#)
Simocephalus cf. exspinosus (#)
Simocephalus cf. mixtus (#)
Simocephalus cf. mixtus (#)
Hap k (*)
Hap l (*)
Hap h (*)
Hap i (*)
Hap j (*)
Hap k (*)
Hap l (*)
Hap h (*)
Hap i (*)
Hap j (*)
Simocephalus serrulatus (#) Simocephalus serrulatus (#)
D. similoides (*)
D. similoides (*)
D. dubium (*)
D. dubium (*)
100
92
100
97
100
77
99
66
87
100
78
63
69
62
0.02 *: Taiwan +: UK #: Mexico and Guatemala
Simocephalus vetulus (+)
75
95
85
76
99
99
63
62
73
99
99
20 *: Taiwan +: UK #: Mexico and Guatemala
Young et al. – Study of Simocephalus from Taiwan 229
to intraspecific variations. Therefore, we prefer
to treat all morphotypes of Simocephalus sensu
stricto from Taiwan as a single species, S. cf.
vetulus, as the publication time of S. vetulus was
earlier than those of the other 2 species.
COI sequence comparison of S. cf. vetulus
from Taiwan with the European S. vetulus showed
that these were not conspecific (Fig. 6). As no
sequences of S. mixtus or S. vetuloides from the
areas of their primary distribution were available
for comparison, it remains unclear whether the
species found in Taiwan are conspecic with those
species. It is possible that Simocephalus found
in Taiwan is either S. mixtus or S. vetuloides or a
new undescribed species. Future studies should
compare sequences of S. vetulus, S. mixtus, and
S. vetuloides collected from the type locations with
sequences of S. cf. vetulus from Taiwan to verify
its taxonomic status.
According to allozymic studies by Hann
(1995), intraspecific differentiation within S. cf.
vetulus in North America was very slight. North
American and European populations were gene-
tically distinct according to the allozyme data, but
no morphological distinctiveness was identified.
In the past, conspecic populations from different
continents were believed to be widespread
within the Cladocera based on morphological
identifications. An intercontinental distribution of
a species is generally presumed to be a result
of passive transport by migratory birds or other
dispersal mechanisms (Dumont and Negrea 2002,
Adamowicz et al. 2009). The alternative hypothesis
of geographical isolation assumes that gene flow
among populations of cosmopolitan species on
different continents is interrupted, and therefore
the question is how large their genetic divergence
is relative to the geographical dis-continuum
scale. For example, Xu et al. (2009) explored the
global phylogeography of the non-cosmopolitan
freshwater cladoceran Polyphemus pediculus
(Linnaeus, 1761) (Crustacea, Onychopoda) using
2 mitochondrial genes, COI and 16s ribosomal (r)
RNA, and 1 nuclear marker, 18s rRNA. The P.
pediculus complex represents an assemblage of at
least 9 largely allopatric, cryptic species. The Far
East harbors exceptionally high levels of genetic
diversity at both the regional and local scales.
In contrast, little genetic subdivision is apparent
across the formerly glaciated regions of Europe
and North America.
Similar to Xu et al. (2009) and many other
previous studies on cosmopolitan cladoceran
species (Ishida et al. 2006, Rowe et al. 2007,
Belyaeva and Taylor 2009, Abreu et al. 2010), our
results indicate that S. cf. vetulus from Taiwan
is probably not the same species as S. vetulus
from the UK, and S. serrulatus from Taiwan is not
conspecific with S. cf. serrulatus from Mexico.
Simocephalus cf. vetulus from Taiwan appears to
be geographically isolated from populations on
other continents. Future studies should collect
barcodes of all morphospecies of Simocephalus
from different locations around the world in order
to reconstruct their systematic relationships.
Acknowledgments: We thank the National Sci-
ence Council of Taiwan for their grant (NSC87-
2311-B-134-001) to support part of this work. We
are very grateful to the anonymous reviewers for
their critical and constructive comments on our
manuscript.
REFERENCES
Abreu MJ, MJ Santos-Wisniewski, O Rocha, TC Orlando.
2010. The use of PCR-RFLP to genetically distinguish
the morphologically close species: Ceriodaphnia dubia
Richard, 1894 and Ceriodaphnia silvestrii Daday, 1902
(Crustacean Cladocera). Braz. J. Biol. 70: 121-124.
Adamowicz SJ, A Petrusek, JK Colbourne, PDN Hebert, JDS
Witt. 2009. The scale of divergence: a phylogenetic
appraisal of intercontinental allopatric speciation in a
passively dispersed freshwater zooplankton genus. Mol.
Phylogen. Evol. 50: 423-436.
Belyaeva M, DJ Taylor. 2009. Cryptic species within the
Chydorus sphaericus species complex (Crustacea:
Cladocera) revealed by molecular markers and sexual
stage morphology. Mol. Phylogen. Evol. 50: 534-546.
Blaxter ML. 2004. The promise of a DNA taxonomy. Phil.
Trans. R. Soc. B Biol. Sci. 359: 669-679.
Boonsom J. 1984. The freshwater zooplankton of Thailand
(Rotifera and Crustacea). Hydrobiologia 113: 223-229.
Chen CS, CH Tzeng, TS Chiu. 2010. Morphological and
molecular analyses reveal separations among
spatiotemporal populations of anchovy (Engraulis
japonicus) in the southern East China Sea. Zool. Stud.
49: 270-282.
Chiang SC, NS Du. 1979. Freshwater Cladocera. Fauna
Sinica. Crustacea. Beijing, China: Science Press,
Academia Sinica, 297 pp.
Costa FO, JR deWarrd, J Boutillier, S Ratnasingham, RT
Dooh, M Hajibabaei, PDN Hebert. 2007. Biological
identifications through DNA barcodes: the case of the
Crustacea. Can. J. Fish. Aquat. Sci. 64: 272-295.
Du NS. 1993. Crustacean biology. Beijing, China: Science
Press, 1003 pp. (in Chinese)
Elias-Gutierrez M, FM Jeronimo, NV Ivanova, M Valdez-
Moreno, PDN Hebert. 2008. DNA barcodes for
Cladocera and Copepoda from Mexico and Guatemala:
highlights and new discoveries. Zootaxa 1893: 1-42.
Dumont HJ, SV Negrea. 2002. Introduction to the class
Branchiopoda. (No. 19) Guides to the identication of the
230 Zoological Studies 51(2): 222-231 (2012)
microinveryebrates of the continental waters of the world.
Leiden, the Netherlands: Backhuys Publishers, 398 pp.
Flössner D. 1972. Krebstiere, Crustacea. Kiemen- und
Blattfüsser, Branchiopoda. Fischläuse, Branchiura.
Tierw. Deutschl. 60. Gustav Fischer Verlag, Jena. 501 p.
(in German)
Folmer O, M Black, W Hoeh, R Lutz, R Vrijenhoek. 1994. DNA
primers for amplification of mitochondrial cytochrome c
oxidase subunit I from diverse metazoan invertebrates.
Mol. Mar. Biol. Biotechnol. 3: 294-299.
Fryer G. 1957. Freeliving freshwater Crustacea from Lake
Nyassa and adjoining waters. Part II. Cladocera and
Conchostraca. Arch. Hydrobiol. 53: 223-239.
Hall TA. 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/98/
NT. Nucleic Acids. Symp. Ser. 41: 95-98.
Hann BJ. 1995. Genetic variation in Simocephalus
(Anomopoda: Daphniidae) in North America: patterns and
consequences. Hydrobiologia 307: 9-14.
Harding JP. 1961. Some South African Cladocera collected by
Dr. A. D. Harrison. Ann. S. Afr. Mus. 46: 35-46.
Hebert PDN, CA Ball, SL deWaard Jr. 2003. Biological identi-
cations through DNA barcodes. Proc. R. Soc. Lond. B
Biol. Sci. 270: 313-321.
Hebert PDN, EH Penton, JM Burns, DH Janzen, W Hallwachs.
2004. Ten species in one: DNA barcoding reveals cryptic
species in the Neotropical skipper butterfly Astraptes
fulgerator. PNAS 101: 14812-14817.
Hebert PDN, S Ratnasingham, SL deWarrd JR. 2003. Bar-
coding animal life: cytochrome c oxidase subunit 1
divergences among closely related species. Proc. R. Soc.
Lond. B 270(Supplement): 96-99.
Hebert PDN, MY Stoeckle, TS Zemlak, CM Francis. 2004.
Identication of birds through DNA barcodes. Plos Biol. 2:
1657-1663.
Hemsen J. 1952. Ergebnisse Österreichischen Iran Expedition
1949/50. Cladoceren und freilebende Copepoden, usw.
Österr. Akad. Wiss. Mathem.-nat. Kl. Abt. I 161: 585-644.
(in German)
Ishida S, AA Kotov, DJ Taylor. 2006. A new divergent lineage of
Daphnia (Cladocera: Anomopoda) and its morphological
and genetical differentiation from Daphnia curcirostris
Eylmann, 1887. Zool. J. Linn. Soc. 146: 385-405.
Johnson DS. 1953. On some Cladocera from South African
muds. Ann. Mag. Nat. Hist. 12: 923-928.
Kim IH. 1998. Key to the Korean freshwater Cladocera. Kor. J.
Syst. Zool. Spec. Issue 2: 43-65. (in Korean)
Kumar S, K Tamura, M Nei. 2004. MEGA 3: integrated
software for molecular evolutionary genetic analysis
sequence alignment. Bioinformatics 5: 150-163.
Lefébure T, CJ Douady, M Gouy, J Gibert. 2006. Relationships
between morphological taxonomy and molecular
divergence within Crustacea: proposal of a molecular
threshold to help species delimitation. Mol. Phylogen.
Evol. 40: 435-447.
Manuilova EF. 1964. Vetvistousye rakoobraznye fauny SSSR.
Opredeliteli po faune SSSR 88. Moscow and Leningrad:
Nauka, 327 pp. (in Russian)
Mayer E, PD Ashlock. 1991. Principles of systematic zoology,
2nd ed. New York: McGraw-Hill, pp. 42-51.
Michael RG, BK Sharma. 1998. Indian Cladocera: fauna of
Indian and adjacent countries. Shillong, Meghalaya,
India: Department of Zoology, North-Eastern Hill Univ.,
262 pp.
Mizuno T, E Takahashi. 1991. An illustrated guide to freshwater
zooplankton in Japan. Hiroshibacho, Suita-shi, Osaka,
Japan: Hoikusha Publishing, 532 pp.
Moore WS. 1995. Inferring phylogenies from mtDNA variation:
mitochondrial-gene trees versus nuclear-gene trees.
Evolution 49: 718-726.
Negrea S. 1983. Cladocera. Fauna Republicii Socialiste
Romania. Volume IV. Fascicula 12. Cladocera. Editura
Academiei Republicii Romania. Bucuresti, 399p.
Nei M. 1987. Molecular evolutionary genetics. New York:
Columbia Univ. Press.
Ni MH. 2005. Systematic studies of Simocephalus (Cladocera:
Daphnnidae) from Taiwan. Master’s thesis, Department
of Applied Science, National Hsinchu Univ. of Education,
Hsinchu, Taiwan, 69 pp.
Orlova-Bienkowskaja MY. 1998. A revision of the clarocen
Genus Simocephalus (Crustacea: Daphniidae). Bull. Nat.
Hist. Mus. Lond. (Zool.) 64: 1-62.
Orlova-Bienkowskaja MY. 2001. Cladocera: Anomopoda
Daphniidae: genus Simocephalus. Leiden, the Nether-
lands: Backhuys, 130 pp.
Rajapaksa R, CH Fernando. 1982. The Cladocera of Sri Lanka
(Ceylon), with remarks on some species. Hydrobiologia
94: 49-69.
Rowe CL, SJ Adamowicz, PDN Hebert. 2007. Three new
cryptic species of the freshwater zooplankton genus
Holopedium (Crustacea: Branchiopoda: Ctenopoda),
revealed by genetic methods. Zootaxa 1656: 1-49.
Rylov WM. 1930. Cladocera et Copepoda in Abhandlungen
der Pamir-Expedition 1928. II. Zoologie: 105-133.
Saitou N, M Nei. 1987. Neighbor-joining method. Mol. Biol.
Evol. 4: 406-425.
Sharam BK. 1978. A note on freshwater cladocerans from
West Bengal. Bangladesh J. Zool. 6: 139-151.
Sars GO. 1903. On the crustacean fauna of central Asia. Part
II. Appendix. Cladocera. Ann. Mus. Zool. Acad. Sci. St.
Petersburg 8: 157-264.
Sars GO. 1916. The freshwater Entomostraca of Cape
Province (Union of South Africa) Part I. Cladocera. Ann.
S. Afr. Mus. 15: 303-351.
Shi XL, XB Shi. 1996. On the species and distribution of
Simocephalus in Heilongjiang Province, China. Acta
Zootax. Sin. 21: 263-276. (in Chinese)
Tautz D, P Arctander, A Minelli, RH Thomas, AP Vogler. 2003.
A plea for DNA taxonomy. Trends Ecol. Evol. 18: 70-74.
Tuo YS. 2002. Freshwater Cladocera of Taiwan. Master’s
thesis, Institute of Mathematics and Science Education,
Hsinchu Education College, Hsinchu, Taiwan, 60 pp.
Uéno M. 1927. On some freshwater branchiopods from China.
Annot. Zool. Jpn. 11: 157-163.
Uéno M. 1966. Cladocera and Copepoda from Nepal. Jpn. J.
Zool. 26: 95-100.
Werestschagin G. 1923. Notiz über die Süsswasserfauna
des Pamirs. Bull. I`Inst. Hydrobiol. Russie 6: 1-40. (in
German)
Xu S, PDN Hebert, AA Kotov, ME Cristescu. 2009. The non-
cosmopolitanism paradigm of freshwater zooplankton:
insights from the global phylogeography of the predatory
cladoceran Polyphemus pediculus (Crustacea,
Onychopoda). Mol. Ecol. 18: 5161-5179.
Yoon SM, HS Kim. 1987. A systematic study on the freshwater
Cladocera from Korea. Kor. J. Syst. Zool. 3: 175-207. (in
Korean)
Yoon SM, W Kim. 2000. Taxonomic review of the cladoceran
genus Simocephalus (Branchiopoda, Anomopoda,
Daphnidae). Kor. J. Limnol. 33: 152-161.
Young et al. – Study of Simocephalus from Taiwan 231
... En el octavo día, se registró el inicio de la etapa adulta con cuatro hembras que median entre 1,3 y 1,6 mm, esta talla fue más frecuente a partir del décimo día, sin embargo, entre los días 40 y 75 los mismos organismos medían entre 2 y 2,5 mm, y con las mediciones complementarias la talla llegó a 2,7 mm. Por lo tanto, la morfometría registrada en las hembras de este ensayo, plantea posibles subregistros de tamaños por especies aceptadas del género Simocephalus, abriendo la posibilidad del uso de otros parámetros alométricos para la comparación morfológica específica a favor de la sistemática de este género tal como lo desarrollaron Young et al. (2012). ...
... It is worth noting that the beginning of the reproductive stage does not represent the end of growth, as occurs in other taxa. On the eighth day, the beginning of the adult stage was recorded with four females measuring between 1.3 and 1.6 mm, this size was more frequent from the tenth day, however, between days 40 and 75 the same organisms measured between 2 and 2.5 mm, and with the complementary measurements, the size reached 2.7 mm. Thus, the morphometry recorded in the females of this trial raises possible sub-registers of sizes accepted for species of the genus Simocephalus, opening the possibility of the use of other allometric parameters for specific morphological comparison in favor of the systematics of this genus as developed by Young et al. (2012). ...
Article
Full-text available
Se empezó un monocultivo a partir de una hembra partenogenética de la cepa Simocephalus mixtus imarpe sp. nov. IMP-BG-Z021 de la laguna Jitarayoc o Guitarra Yoc en la Región Ayacucho sobre 4600 msnm. Se evaluó el crecimiento diario de una muestra (n= 60) nacidos en un intervalo de 8 horas, el fitness fue alterado al medir los mismos individuos a lo largo de su vida, observándose un comportamiento resistente a la manipulación. Los individuos fueron criados en un beaker de medio litro dentro de una cámara climática a 16 °C, fotoperiodo 14:12 O: L, salinidad 0‰ y fueron alimentados con la microalga Chlorella sp. Se registraron los parámetros longevidad (75 días), progenie (20,4 crías/hembra en promedio), etapa pre reproductiva (8 días), reproductiva y post reproductiva casi ausente. Además, se consideraron los parámetros biométricos longitud y ancho durante el desarrollo de las crías (708,99 µm) hasta su adultez (2063,39 µm) y eclosión de huevos de resistencia o efipio al aumentar 8 °C dela temperatura en la que se desarrolló. La tasa de crecimiento individual fue k= 13,69 y su longitud máxima fue 2750 µm. Se propone la relación de procesos de especiación con las características geológicas del hábitat, así como la conservación de esta cepa como organismos andinos aptos para experimentación y alimento vivo nativo debido a su rusticidad al manejo y adaptación a temperaturas templadas.
... Among Cladocerans, family Daphniidae is more common consisting mainly of Daphnia, Ceriodaphnia and Simocephalus. These members can be helpful in assessment of environmental conditions and can act as good bioindicators of aquatic pollution [2][3][4] . The behavioural change is a manifestation of the motivational, biochemical, physiological and environmentally influenced state of the organism. ...
Article
Full-text available
Intense industrialization in India led to release of toxic effluents in surface water bodies causing deleterious effects on freshwater flora and fauna. Simocephalus vetulus (crustacean-cladocera) a tailless fresh water flea is an important member of the zooplankton community and base of the aquatic food chain of freshwater aquatic ecosystems. Fresh water tailless flea, S. vetulus exposed to 5%, 10%, 15% and 20% of SIDCUL Integrated Industrial Estate (SIDCUL-IIE) effluent of Haridwar showed marked changes in behaviour like phototaxis, geotaxis, avoidance activity, appendage movements, swimming, feeding as well as in the morphology, specially in cuticular coloration. The exposed fleas showed hyper activity at initial stage followed by erratic swimming, loss of balance, darkening of carapace, loss of aggressive behaviour and mucous secretion. The food detection and consumption of the fleas was found normal in the initial stage which considerably declined after 48, 72 and 96 hrs exposure of SIDCUL IIE effluent. The phototaxis geotaxis and avoidance indices were found decreased after exposure to 5%, 10%, 15% and 20% SIDCUL IIE effluent, as observed after 7, 14 and 21 days exposure. In the present study it was clearly evident that the intensity of effects was dose and duration dependent. The findings of present study are beneficial for prediction of safe concentration of industrial effluents to be released for well being of freshwater aquatic bodies. The behavioural parameters of the tailless fresh water flea may serve as better biomarkers in relation to industrial effluent toxicity. The role of S. vetulus is significant in environmental monitoring has been discussed.
... The specimens were examined under a Leica DM 5000 camera attached to a microscope fitted with an image analyzer, and then identified following Smirnov (1971Smirnov ( , 1996, Michael and Sharma (1988), Subhash Babu and Nayar (2004); Kotov et al. (2012Kotov et al. ( , 2013 and Pascual et al. (2014). We used specialized papers to aid in the identification of species of Macrothrix (Dumont et al., 2002;Kotov, 2008), Kurzia (Hudec, 2000;Padhye and Van Damme, 2015), Celsinotum (Rajapaksa and Fernando, 1985), Chydorus (Rajapaksa and Fernando, 1986a;Smirnov, 1996;Van Damme and Dumont, 2007), Alona (Sinev, 1999;, Karualona (Dumont and Silva-Briano, 2000;, Coronatella (Van Damme and Sousa et al., 2015a), Notoalona (Rajapaksa and Fernando, 1987), Anthalona (Van Damme et al., 2011;Sinev and Kotov, 2012;Sousa et al., 2015b), Dadaya (Rajapaksa and Fernando, 1982a), Diaphanosoma (Korovchinsky, 1998(Korovchinsky, , 2002, Pleuroxus (Hudec and Illyova, 1998;Chiambang and Dumont, 2004;Sinev and Sanoamuang, 2013), Simocephalus (Young et al., 2012), Bosmina (Taylor et al., 2002;Kotov et al., 2009), Bosminopsis (Rey and Vasquez, 1986;Kotov, 1999), and Ephemeroporus (Frey, 1982b;Smirnov, 1996). The specimens were deposited in the Laboratory of Fisheries and Aquatic Ecology of the Rajiv Gandhi University, Itanagar, India. ...
Article
The plankton samples collected from the Subansiri floodplain wetlands revealed a rich Cladocera assemblage of 55 species belonging to 30 genera and 7 families. The species richness represents 42% and 75% of the total amount of fresh water species reported from India and Assam, respectively. Chydoridae was the most speciose family, with 31 species, while Ilyocryptidae was represented by a single species. Sididae, Daphniidae, Bosminidae, Moinidae and Macrothricidae were represented by four, five, three, two and four species, respectively. The faunal composition is represented by cosmopolitan, tropical and oriental elements. The documentation of Diaphanosoma dubium, Latonopsis australis, Simocephalus mixtus, Chydorus sphaericus, Chydorus parvus, Chydorus ovalis, Alonella clathratula, Pleuroxus cf. denticulatus, Picripleuroxus quasidenticulatus, Celsinotum macronyx, Coronatella anodonta and Kurzia (Rostrukurzia) brevilabris has biogeographic importance. We provide brief geographical distributional remarks about these 12 species from the collected samples. This was a preliminary study, as the fauna from the Indian subcontinent is poorly documented, and requires a taxonomic revision as a whole. The faunistic diversity of cladocerans comprises a clear representation of a tropical cladoceran assemblage.
... The specimens were examined under a Leica DM 5000 camera attached to a microscope fitted with an image analyzer, and then identified following Smirnov (1971Smirnov ( , 1996, Michael and Sharma (1988), Subhash Babu and Nayar (2004); Kotov et al. (2012Kotov et al. ( , 2013 and Pascual et al. (2014). We used specialized papers to aid in the identification of species of Macrothrix (Dumont et al., 2002;Kotov, 2008), Kurzia (Hudec, 2000;Padhye and Van Damme, 2015), Celsinotum (Rajapaksa and Fernando, 1985), Chydorus (Rajapaksa and Fernando, 1986a;Smirnov, 1996;Van Damme and Dumont, 2007), Alona (Sinev, 1999;, Karualona (Dumont and Silva-Briano, 2000;, Coronatella (Van Damme and Sousa et al., 2015a), Notoalona (Rajapaksa and Fernando, 1987), Anthalona (Van Damme et al., 2011;Sinev and Kotov, 2012;Sousa et al., 2015b), Dadaya (Rajapaksa and Fernando, 1982a), Diaphanosoma (Korovchinsky, 1998(Korovchinsky, , 2002, Pleuroxus (Hudec and Illyova, 1998;Chiambang and Dumont, 2004;Sinev and Sanoamuang, 2013), Simocephalus (Young et al., 2012), Bosmina (Taylor et al., 2002;Kotov et al., 2009), Bosminopsis (Rey and Vasquez, 1986;Kotov, 1999), and Ephemeroporus (Frey, 1982b;Smirnov, 1996). The specimens were deposited in the Laboratory of Fisheries and Aquatic Ecology of the Rajiv Gandhi University, Itanagar, India. ...
Article
Full-text available
The plankton samples collected from the Subansiri floodplain wetlands revealed a rich Cladocera assemblage of 55 species belonging to 30 genera and 7 families. The species richness represents 42% and 75% of the total amount of fresh water species reported from India and Assam, respectively. Chydoridae was the most speciose family, with 31 species, while Ilyocryptidae was represented by a single species. Sididae, Daphniidae, Bosminidae, Moinidae and Macrothricidae were represented by four, five, three, two and four species, respectively. The faunal composition is represented by cosmopolitan, tropical and oriental elements. The documentation of Diaphanosoma dubium, Latonopsis australis, Simocephalus mixtus, Chydorus sphaericus, Chydorus parvus, Chydorus ovalis, Alonella clathratula, Pleuroxus cf. denticulatus, Picripleuroxus quasidenticulatus, Celsinotum macronyx, Coronatella anodonta and Kurzia (Rostrukurzia) brevilabris has biogeographic importance. We provide brief geographical distributional remarks about these 12 species from the collected samples. This was a preliminary study, as the fauna from the Indian subcontinent is poorly documented, and requires a taxonomic revision as a whole. The faunistic diversity of cladocerans comprises a clear representation of a tropical cladoceran assemblage.
... These members can be helpful in assessment of environmental conditions and can act as better bioindicators of aquatic pollution. (Rao, 2001;Raghunathan and Kumar, 2003;Lee et al., 2004;Young, et al., 2012 andMishra, et al., 2016). The behavioural change is a manifestation of the motivational, biochemical, physiological and environmentally influenced state of the organism. ...
Article
Full-text available
Water fleas constitute major zooplankton population of fresh water aquatic ecosystem. Their population density is an indicative of well-being of aquatic bodies. Simocephalus vetulus (Crustacea-Cladocera) is a tailless water flea and is well suited lab model for environmental monitoring. Copper a Gray listed heavy metals despite being an essential micronutrient, becomes highly toxic when present in excess quantity in aquatic ecosystem thereby causing deleterious effects on aquatic flora as well as fauna. The water-flea exposed to acute 0.37 mg/l (96hr LC 50), sub-acute 0.0925 mg/l (25% of 96hr LC 50) value and chronic 0.037 mg/l (10% of 96hr LC 50) value of copper sulphate and acute 0.16 mg/l (96 hr LC 50), sub-acute 0.04 mg/l (25% of 96 hr LC 50) value and chronic 0.016 mg/l (10% of 96 hr LC 50) value of potassium chromate showed behavioural alterations like initial hyperactivity, fast appendage movements and in phototaxis, geotaxis and avoidance indices. At later stage erratic swimming and spinning, reduced activity, loss of balance, reduced feeding and darkening of cuticular coloration, reduced phototactic, geotaxis and avoidance indices were the major effects on its behaviour. The behavioural alterations of S. vetulus showed the most susceptible and foremost indication of potential toxic effects. Various behavioural parameters, used in present study may serve as better biomarkers about metal toxicity and monitoring of drinking water quality.
... KX442671-KX442686, Popova et al. 2016) and from Taiwan (as D. similoides, GenBank accession nos. AB549199-AB549200, Young et al. 2012). Based on this evidence, we currently consider D. sinensis to be more appropriate nomenclature than D. carinata for the D. similis-like taxon sequenced in the present study, even though the identity of the reference sequence was D. carinata (Table 1). ...
Article
Although DNA barcoding is a promising tool for the identification of organisms, it requires the development of a specific reference sequence library for sample application. In the present study we developed a Lake Kasumigaura, Japan, zooplankton DNA barcode library to increase the sensitivity of future zooplankton monitoring for detecting lake ecosystem condition changes. Specifically, the mitochondrial cytochrome c oxidase subunit I (mtCOI) haplotype, i.e., the primary DNA barcode, was examined for each zooplankton taxon. In crustaceans, 37 mtCOI haplotypes were obtained from 99 individuals, representing four and 15 morpho-species of Copepoda and Cladocera, respectively. Comparing these sequences with those in GenBank shows that the lake harbors putative non-indigenous species, such as Daphnia ambigua. In rotifers, 132 mtCOI haplotypes were obtained from 302 individuals, representing 11 genera and one unclassified taxon. The automatic barcode gap discovery (ABGD) algorithm separated these haplotypes into 43 species. Brachionus cf. calyciflorus was divided into five ABGD species, and different ABGD species tended to occur in different seasons. Seasonal ABGD-species succession was also observed within Polyarthra spp. and Synchaeta spp. These seasonal successions were not detected by inspections of external morphology alone. Accepting up to 7% sequence divergence within the same species, mtCOI reference sequences were available in GenBank for three, 13, and 17 species in Copepoda, Cladocera, and Rotifera, respectively. The present results, therefore, reveal the serious shortage of mtCOI reference sequences for rotifers, and underscore the urgency of developing rotifer mtCOI barcode libraries on a global scale.
... "similis" is a separate taxon related to D. exilis; and a novel mitochondrial lineage related to that of D. similis was revealed from a single locality in Germany (Petrusek 2003;Colbourne et al. 2006;Adamowicz et al. 2009). Young et al. (2012) assigned a genetic lineage from Taiwan to Daphnia similoides (without comparison with material to the type locality), and Ma et al. (2016) followed the same approach when dealing with Chinese populations (assigning them to D. similoides sinensis Gu, Xu, Li, Dumont et Han, 2013). However, morphological and genetic evidence is most informative in resolving Daphnia species groups when presented in a coordinated effort (Kotov 2015). ...
Article
Full-text available
Species of the genus Daphnia O.F. Müller, 1785 (Cladocera: Daphniidae) have become very important models in evolu-tionary biology research. Previous morphological and genetic evidence suggests that numerous closely related “species groups” exist within the subgenus Daphnia (Ctenodaphnia) Dybowski & Grochowski, 1895, containing both described and undescribed species. The Daphnia similis group is among these species groups. The aim of the present paper is to revise the taxonomy of the Daphnia (Ctenodaphnia) similis group in the Old World with both morphological and genetic evidence (based on mitochondrial COI and 12S rRNA genes). We found that there are at least four species in the Old World D. similis species group: D. similis Claus, 1876; D. sinensis Gu, Xu, Li, Dumont et Han, 2013; D. similoides Hudec, 1991 and D. inopinata sp. nov. These four taxa of the similis-group, confused previously with D. similis, have different distri-butional ranges in the Old World, from extremely wide, spanning several biogegraphic regions (as D. sinensis), to regional endemics (D. similoides) and even species known so far from a single locality (D. inopinata sp. nov.). The Daphnia similis group provides another example in the cladocerans whereby the study of males yields more valuable characters for tax-onomy than the study of parthenogenetic females.
Article
Full-text available
We investigated microcrustaceans inhabiting arsenic contaminated and non-contaminated freshwater to identify potential bioindicators of arsenic contamination in the tropical freshwater of Matehuala in northern Mexico. We collected water, sediment, and zooplankton, at five sampling points during three sampling campaigns. We determined water temperature, pH, electrical conductivity, dissolved oxygen, alkalinity, salinity, and total arsenic concentration in water. Additionally, we determined total arsenic and arsenic speciation in sediment samples. We identified microcrustaceans and determined abundance, richness, and Shannon Index. We also investigated relationships and correlations between physiochemical and ecological variables. Results showed that arsenic concentrations in freshwater ranged from 0.001 to 53.23 mg/L, while total arsenic in sediments ranged from 10.37 to 2472.84 mg/kg as As + 5. Six microcrustacean species were found in highly and moderately contaminated water (Latonopsis australis, Eucyclops chihuahuensis, Acanthocyclops americanus, Pleuroxus (Picripleuroxus) quasidenticulatus, Macrocyclops albidus, and Paracyclops chiltoni), while five species were found in arsenic-free water (Simocephalus punctatus, Alona glabra, Eucyclops leptacanthus, M. albidus, and P. quasidenticulatus). An inverse relationship was observed between microcrustacean richness and arsenic. However, the scope of the data did not allow for a strong and significant correlation. Nevertheless, among the species inhabiting As-free water, S. punctatus showed potential to be further tested as a bioindicator of As contamination in Matehuala. Identification of potential bioindicators could help monitor water quality and increase understanding of the incorporation and toxicity of As in freshwater-sensitive and freshwater-metallotolerant microcrustaceans, which, in turn, might help us to understand As incorporation in the food web.
Chapter
Zooplankton species are distributed throughout the three-dimensional water body and most species are motile, which means that their distribution (vertically and horizontally) is variable and tends to be patchy (Tonolli 1971b; Levin and Segel 1976; Vilar et al. 2003). The term “patchiness” was first introduced by Hardy in 1936, but the concept was first described by Haeckel in 1890 with the observation that plankton was not “evenly” distributed (Cassie 1968). Various species have different environmental preferences and occupy different habitats, which along with water movements produces spatial and temporal heterogeneity. The various methods of collecting and counting zooplankton can lead to gear- and method-specific biases (Mack et al. 2012). Therefore, quantitative sampling and counting are difficult. Errors associated with quantitative research may be categorized into three types. Errors associated with (1) counting the specimens in a sample, errors associated with (2) obtaining a representative sample of the population in a site and water body (Wetzel and Likens 1990), and errors associated with the proper (3) species identification. We will deal with these errors and their solutions more deeply in the following subchapters.
Chapter
This section covers description of Cladocera taxonomic ranks and provides a list of genera and species reported from Europe.
Article
Full-text available
Molecular approaches have greatly advanced our understanding of species diversity and biogeography in the cladoceran crustaceans. Here, we provide the first large-scale examination of taxonomic diversity in the genus Holopedium Zaddach, 1855, by characterizing patterns of allozyme, mtDNA, and morphological variation from a total of 193 sites from three continents, including collections from near the type localities for the two generally recognized species, Holopedium gibberum Zaddach, 1855, and Holopedium amazonicum Stingelin, 1904. Allozyme data were only available for North American samples but revealed the presence of four species. Divergence patterns in the mitochondrial cytochrome c oxidase subunit I (COI) gene supported those species, as well as a fifth taxon endemic to South America. The five putative species are separated by substantial sequence (8.7–24.5%) and allozyme (0.36–1.54 Nei’s distance) divergences, while intraspecific genetic diversity was generally limited in comparison. Although two of these species exhibited little morphological differentiation from their closest relatives, and diagnostic traits were not found among the characters considered, a population-level approach revealed significant morphological differences among all pairs of taxa. We therefore present both an allozyme key and a morphological/geographic key to all species, as well as new or augmented descriptions for all five species. H. gibberum s.s. is distributed in Europe and across arctic North America, while its cryptic sister species, H. glacialis n. sp., is widely distributed across temperate North America. H. amazonicum s.s. is apparently restricted to the Amazon basin, H. atlanticum n. sp. occurs in lakes along the eastern margin of North America, while H. acidophilum n. sp. occurs sporadically across North America along a narrow band of middle latitudes. Due to high morphological variability within species, as well as the detection of cryptic diversity, we suggest that genetic analyses should be performed on populations from other geographic regions and should always accompany the recognition of new species of Holopedium.
Article
Full-text available
DNA barcoding, based on sequence diversity in the mitochondrial COI gene, has proven an excellent tool for identifying species in many animal groups. Here, we report the first barcode studies for freshwater zooplankton from Mexico and Guatemala and discuss the taxonomic and biological implications of this work. Our studies examined 61 species of Cla-docera and 21 of Copepoda, about 40% of the known fauna in this region. Sequence divergences among conspecific individuals of cladocerans and copepods averaged 0.82% and 0.79%, respectively, while sequence divergences among congeneric taxa were on average 15-20 times as high. Barcodes were successful in discriminating all species in our study, but sequences for Mexican Daphnia exilis overlapped with those of D. spinulata from Argentina. Our barcode data revealed evidence of many species overlooked by current classification systems -for example, based on COI genotypes the Diapahanosoma birgei group appears to include 5 species, while Ceriodaphnia cf. rigaudi, Moina cf. inicrura, Mastigodiaptomus albuquerquensis and Mastigodiaptomus reidae all include 2-3 taxa. The barcode results support recent taxonomic revisions, such as recognition of the genus Lebenis, and the presence of several species in the D. birgei and Chydorus sphaericus complexes. The present results indicate that DNA barcoding will provide powerful new insights into both the incidence of cryptic species and a better understanding of zooplankton distributions, aiding evaluation of the factors influencing competitive outcomes, and the colonization of aquatic environments.
Article
Full-text available
Molecular approaches have greatly advanced our understanding of species diversity and biogeography in the cladoceran crustaceans. Here, we provide the first large-scale examination of taxonomic diversity in the genus Holopedium Zaddach, 1855, by characterizing patterns of allozyme, mtDNA, and morphological variation from a total of 193 sites from three continents, including collections from near the type localities for the two generally recognized species, Holopedium gibberum Zaddach, 1855, and Holopedium amazonicum Stingelin, 1904. Allozyme data were only available for North American samples but revealed the presence of four species. Divergence patterns in the mitochondrial cytochrome c oxidase subunit I (COI) gene supported those species, as well as a fifth taxon endemic to South America. The five putative species are separated by substantial sequence (8.7-24.5%) and allozyme (0.36-1.54 Nei's distance) divergences, while intraspecific genetic diversity was generally limited in comparison. Although two of these species exhibited little morphological differentiation from their closest relatives, and diagnostic traits were not found among the characters considered, a population-level approach revealed significant morphological differences among all pairs of taxa. We therefore present both an allozyme key and a morphological/geographic key to all species, as well as new or augmented descriptions for all five species. H. gibberum s.s. is distributed in Europe and across arctic North America, while its cryptic sister species, H. glacialis n. sp., is widely distributed across temperate North America. H. amazonicum s.s. is apparently restricted to the Amazon basin, H. atlanticum n. sp. occurs in lakes along the eastern margin of North America, while H. acidophilum n. sp. occurs sporadically across North America along a narrow band of middle latitudes. Due to high mor-phological variability within species, as well as the detection of cryptic diversity, we suggest that genetic analyses should be performed on populations from other geographic regions and should always accompany the recognition of new spe-cies of Holopedium.
Article
Full-text available
With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
Article
An accurately resolved gene tree may not be congruent with the species tree because of lineage sorting of ancestral polymorphisms. DNA sequences from the mitochondrially encoded genes (mtDNA) are attractive sources of characters for estimating the phylogenies of recently evolved taxa because mtDNA evolves rapidly, but its utility is limited because the mitochondrial genes are inherited as a single linkage group (haplotype) and provide only one independent estimate of the species tree. In contrast, a set of nuclear genes can be selected from distinct chromosomes, such that each gene tree provides an independent estimate of the species tree. Another aspect of the gene-tree versus species-tree problem, however, favors the use of mtDNA for inferring species trees. For a three-species segment of a phylogeny, the branching order of a gene tree will correspond to that of the species tree if coalescence of the alleles or haplotypes occurred in the internode between the first and second bifurcation. From neutral theory, it is apparent that the probability of coalescence increases as effective population size decreases. Because the mitochondrial genome is maternally inherited and effectively haploid, its effective population size is one-fourth that of a nuclear-autosomal gene. Thus, the mitochondrial-haplotype tree has a substantially higher probability of accurately tracking a short internode than does a nuclear-autosomal-gene tree. When an internode is sufficiently long that the probability that the mitochondrial-haplotype tree will be congruent with the species tree is 0.95, the probability that a nuclear-autosomalgene tree will be congruent is only 0.62. If each of k independently sampled nuclear-gene trees has a probability of congruence with the species tree of 0.62, then a sample of 16 such trees would be required to be as confident of the inference based on the mitochondrial-haplotype tree. A survey of mtDNA-haplotype diversity in 34 species of birds indicates that coalescence is generally very recent, which suggests that coalescence times are typically much shorter than internodal branch lengths of the species tree, and that sorting of mtDNA lineages is not likely to confound the species tree. Hybridization resulting in transfer of mtDNA haplotypes among branches could also result in a haplotype tree that is incongruent with the species tree; if undetected, this could confound the species tree. However, hybridization is usually easy to detect and should be incorporated in the historical narrative of the group, because reticulation, as well as cladistic events, contributed to the evolution of the group.