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Morphological, morphometrical and molecular (CO1 and ITS) analysis of the

rotifer Asplanchna brightwellii from selected freshwater bodies in Central
Mexico

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Morphological, morphometrical and molecular (CO1 and ITS)

analysis of the rotifer Asplanchna brightwellii from selected
freshwater bodies in Central Mexico
Jorge Jiménez-Contreras1, S.S.S. Sarma2,* Elías Piedra-Ibarra3,
Marissa Calderón-Torres4 and S. Nandini2
1
Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Av. Universidad
3000 Coyoacán, Mexico City, 04510, Mexico
2
Laboratorio de Zoología Acuática, Edificio Unidad de Morfofisiología, Universidad Nacional Autónoma de México,
Campus Iztacala, Av. de los Barrios no. 1, Iztacala, Tlalnepantla, Edo. de México, 54090, Mexico
3
Unidad de Biomedicina, Universidad Nacional Autónoma de México Campus Iztacala, Av. de los Barrios no. 1, Iztacala,
Tlalnepantla, Edo. de México, 54090, Mexico
4
Unidad de Prototipos, Universidad Nacional Autónoma de México Campus Iztacala, Av. de los Barrios no. 1, Iztacala,
Tlalnepantla, Edo. de México, 54090, Mexico
*Corresponding Author email : ssssarma@gmail.com

Abstract
We evaluated different strains of the rotifer Asplanchna brightwellii collected from central Mexico
using morphology, morphometry and molecular tools (CO1 and ITS). Three distinct clonal populations
from each of the 3 regions (Mexico City, State of Mexico and State of Guerrero) were established under
Publication Info
laboratory conditions. For a given waterbody, morphometric comparisons within the populations of
A. brightwellii showed almost stable measurements of trophi and with no statistically significant
Paper received:
differences among them (p>0.05). However, asplanchnid body length and width as well as the cyst
30 August 2012 diametervaried significantly depending on the waterbody from which A. brightwellii was collected.
The smallest adults (about 700 µm) were from Valerio Trujano lake (Guerrero State) samples while the
Revised received: largest were from Xochimilco lake. Similar tendencies were reflected in the diameter of resting eggs.
09 January 2013 In addition, morphologically the cysts of A. brightwellii from the three waterbodies showed slightly
different pattern. The number of globular structures on the surface of cysts was smaller for Valerio
Accepted: Trujano strain, while these were larger and less numerous for both Xochimilco and Zumpango strains.
01 February 2013 The ITS region tree displayed two groups Xochimilco and Valerio Trujano ­Zumpango, this analysis did
not reflect the morphological grouping; on the contrary the CO1 gene tree separated the populations
according to morphological clusters and location (Xochimilco, Valerio Trujano and Zumpango lakes).
When the tree was built using the combination of both ITS and CO1 sequences, the phylogenetic
relationships observed on CO1 gene were consistent; but showed differences with the relationships
observed on ITS region tree (only two groups).

Key words
CO1, Fingerprinting, ITS, Resting eggs, Rotifers, Trophi

Introduction has been successfully used in the separation of many

species of rotifers, it is not useful to distinguish cryptic
Rotifers are common in a variety of freshwater, species or for same species thatexhibits cyclomorphosis
brackish, saline and marine water habitats (Wallace et al., which also happens for the rotifer genus Asplanchna (José
2006). The most important characteristic for the entire phylum de Paggi, 2002).
is the presence of muscular pharynx (mastax) with chitinous
jaws (trophi) in females. Usually the identification of many The family Asplanchnidae comprises of three genera
rotifers species is based on the morphology of trophi alone Harringia benthic organisms and with a foot well
(Fontaneto et al., 2008). Although this morphological trait developed, Asplanchnopus semi-planktonic with reduced

© Triveni Enterprises, Lucknow (India ) Journal of Environmental Biology, Vol. 34, 1039-1046, November 2013

Journal of Environmental Biology, November 2013

1040 J.J. Contreras et al.

foot and Asplanchna planktonic without foot (Walsh et al., from molecular data and that derived from morphological
2005). Within the genus Asplanchna there are nine species analysis. The molecular analysis includes scrutiny of large
which are distributed from temperate to tropical regions and small subunits of ribosomal RNA genes (rRNA),
(Segers, 2007). The general body shape of Asplanchna is mitochondrial DNA, mainly the cytochrome oxidase c
not very useful for identification. Therefore trophi is largely subunit 1 gene (CO1) and sequence analysis of the internal
used for distinguishing different species. However, currently transcribed spacer (ITS) regions of the rRNA genes (Cooke
there are still many problems related to taxonomy of trophi and Duncan, 1997).
in the genus Asplanchna, because the differences among
the closely related species (e.g., A. brightwellii and A. The CO1 gene has been chosen as DNA barcoding
sieboldii) are difficult to describe (Altindag et al., 2009; of eukaryotes. It codifies for a mitochondrial protein, which
Chang et al., 2010). In addition, within a given species of is important in the electron transport chain, and therefore
Asplanchna the morphological variations in trophi of field- has a central role in the metabolism of eukaryotic aerobic
collected specimens are large and can lead to mistakes in organisms. In addition, mitochondrial genes have high
identifying the species.For example, the trophi size of A. evolutionary rates than nuclear genes (Strüder-Kypke and
sieboldii can vary from 130-170 µm and the tooth on the Lynn,2010). However,CO1 gene has limited use for
inner margin of rami also varies considerably (José de Paggi, identifying certain taxonomic groups, such as fungi, with
low amounts of variation in the genes and also by the
2002). In addition, A. sieboldii develops different morphs
variable structure of the mitochondrial genomes. Therefore,
depending on the availability of food in the environment
ITS has been established as a barcode for fungi and thought
(Chang et al., 2010). If the available prey items are large
be a more suitable DNA barcoding for plants (Chen et al.,
(such as cladocerans or copepods) then the large
2010).
campanulate forms appear in the population;when the food
availability is in short, to avoid cannibalism, some individuals The aim of this work was to understand whether
in the population develop lateral humps (Wallace et al., intra- and inter-populational variability of A. brightwellii
2006). Morphometric analysis of clonal cultures of a given from geographically isolated waterbodies in three States of
species of Asplanchna is helpful to quantify the range of Central Mexico indicate the possible existence of cryptic
variation in the morphology of trophi. This approach too diversity. For this, data of trophi measurements, structure
has limitations because establishing clonal cultures of of resting eggs and CO1 gene analysis were used. We also
widely separated geographic strains is both expensive and analyzed the ITS sequence as a complementary to barcode
regulatory national laws often prevent introduction of foreign and thus we compared the results of ITS with the CO1 gene
taxa. The establishment of cultures of clones of species classification.
obtained from within a nation is thus helpful for
morphometric analysis of different stages in the life history. Materials and Methods
However, this alone is not sufficient for species that show
Live zooplankton samples from different zones
cryptic diversity. Therefore, it is necessary to employ other
(pelagic and littoral or near shore) of the three freshwater
biological tools such as molecular analysis (Schröder and
bodies viz., Xochimilco Nativitas, (Mexico City), Zumpango
Walsh, 2010).
(State of Mexico), Valerio Trujano (Guerrero State) were
Temperature is one the important natural variables separately collected using a plankton net of 50 µm mesh
affecting the morphology, distribution and density of rotifers size (Table 1). All the chosen waterbodies are within the
(Wallace and Snell, 2010).Factors other than temperature national territorial limits of Mexico, and differ by a distance
too have considerable influence on the morphological traits of about 500 km. Live samples were brought to the laboratory
of rotifers. In addition to the well-known phenomenon of and observed using a Nikon stereomicroscope SMZ645.
cyclomorphosis that occurs in various rotifer genera Selected physico-chemical variables of water were also
including Asplanchna, factors such as topographical, measured from the site at the time of sampling. From the live
hydrological and altitude offer opportunities for the zooplankton samples, we isolated between 10 and 20
development of isolated populations (Wallace et al., 2006). individuals of Asplanchna spp., and transferred them
This aspect has not been well studied for many rotifer genera individually into100 ml glass beakers containing 50 ml of
except for Brachionus (Ortells et al., 2006). Genetic traits of moderately hardwater, the EPA medium. The EPA medium
isolated populations are also dependent on physical barriers was daily prepared by dissolving 1.14 mM NaHCO3, 0.44
(Hoffmann and Rieseberg, 2008). mM CaSO4, 0.50 mM MgSO4 and 54 mM KCl in 1 l of distilled
water (Weber, 1993). In order to feed Asplanchna, we
Recently molecular analysis has been considered as separately cultured three brachionid rotifers, viz. Brachionus
a valuable tool for phylogenetic studies where it has been havanaensis, B. rubens and Plationus patulus using the
demonstrated the congruence between taxonomy derived single celled green alga Chlorella vulgaris as the diet. C.

Journal of Environmental Biology, November 2013

Morphological and molecular analysis of Asplanchna 1041

vulgaris was batch-cultured in 2 l transparent bottles using

Bold’s basal medium (Borowitzka and Borowitzka, 1988).
Alga in the log-phase of growth was harvested, centrifuged
at 300 rpm for 5 min., rinsed and re-suspended in distilled
water. Form the algal concentrate we quantified cell density
using a haemocytometer and offered it to brachionid prey
at 0.5X106 cells ml-1 after diluting as needed using EPA
medium. The other test conditions were: temperature 23±1°C,
pH 6.8-7.2, continuous but diffused fluorescent illumination,
the medium and the prey daily replaced.

Individuals of Asplanchna from each jar were offered

the prey (Brachionus havanaensis, B. rubens and Plationus
patulus in equal proportions) at a density of about 5 ind. ml-1.
In order to keep the prey active, we added low density of
Chlorella (at 0.05X106 cells ml-1 which is not enough for the
brachionids to reproduce (Nandini et al., 1997). When the
asplanchnid density was high in each jar (about 1 ind. ml-1),
a few individuals from each jar were separated using a
Pasteur pipette and transferred to a compound microscope
and identified using José de Paggi (2002). Trophi were
separated using commercial sodium hypochlorate solution
(8%). After ascertaining which culture jars contained A.
brightwellii, rest of the jars were discarded. We used
individuals of A. brightwellii from different zones of a given and populations of A. brightwellii. OR= Outer margin of the ramus,
waterbody and were considered here as distinct IR = Inner margin of the ramus, S = spine on the inner margin of
populations. Populations of A. brightwellii from the three ramus and OB = Outer margin of bulla.
different geographical regions were treated as distinct
clones. isolate trophi. Then by orienting the trophi at appropriate
Morphological and morphometric analyses : To perform position, they were drawn using camera lucida (at high
these analyses individuals of different clones (and their magnification, 100X). The measurements of the trophi were
populations) of A. brightwellii collected from each of the done using Digital Plan Measure (Scale Master Pro, Model
three waterbodies indicated earlier were used. These were 6025). We measured the inner and outer margins of the ramus
separately cultured as monoclonal cultures starting with and averaged them; the same procedure was done for the
one amictic female (three independent populations per site). bulla, as shown in Fig. 1. From each clone about 15
For the morphometry of trophi adults were used. individuals were measured and thus resulting into 40-45
Measurements were done for the ramus, the tooth on inner specimens per study site.
margin of the ramus and the bulla (Fig. 1). These were done
For electron microscopy preparations, the trophi
using both optical and electron microscopy. For obtaining
were rinsed several times using distilled water and mounted
cysts, the populations were allowed to grow further until
density-dependent sexual reproduction produced males and dry on stubs for cold coating following De Smet (1998). The
resting eggs (Wallace and Snell, 2010). Cysts thus collected resting eggs were air-dried for 24hr and later mounted on
from the culture jars were employed for both light and stubs for cold coating. We used scanning electron
electron microscopy studies; morphometric measurements microscope (JEOL, LV 5900) at 10,000–25,000X for
included cyst diameter while morphological description morphological observations.
consisted of characteristic pattern of ornamentation on cyst
DNA amplification and sequence analysis : Since
surface.
Asplanchna was fed brachionids, the gut contained DNA
For light microscopic observations, we first drew of prey. This was eliminated by starving the predators for 6-
the length and breadth of individuals using a light 12 hr by which time no prey items were present in the gut
microscope (at low magnification, 10-20X) fitted with and the previously consumed brachionids were fully
calibrated camera lucida. Because Asplanchna is an illoricate, digested (Nandini and Sarma, 1999). Genomic DNA of starved
it was measured in live condition. Subsequently, the adult A. brightwellii was extracted by alkaline-boiling lysis.
specimens of A. brightwellii were individually used to PCR amplifications were performed with Taq DNA

Journal of Environmental Biology, November 2013

1042 J.J. Contreras et al.

polymerase (Invitrogen) using specific primers for a large spine on the inner margin of the ramus.Information
mitochondrial CO1 gene HCO2198: 5´TAA ACT TCA GGG on the morphometry of A. brightwellii obtained from
TGA CCA AAA AAT CA 3´ : 5´TGA TTT TTT GGT CAC different waterbodies showed significant differences in the
CCT GAA GTT TA 3´and for nuclear ITS region (ITS4: body size and the trophi size (Table 2).For a given waterbody,
TCCTCCGCTATTGATATGC, ITS5: GGAAGTAAAAG comparisons within the three populations of A. brightwellii
TCGTAACAAGG). For the PCR amplification we used the showed almost stable measurements of trophi and with no
following program: 1 cycle for 2 min at 94 °C, 30 cycles for 30 statistically significant differences among them (p>0.05, one
sec at 94 °C, 30 sec at 57° C annealing temperature of ITS way ANOVA, Table 3). However, asplanchnid body length
region, 1 min at 72°C; and 1 cycle for 5 min at 72°C; for CO1 and width varied significantly depending on the waterbody
amplification were 40 cycles with a annealing temperature from which A. brightwellii was collected. In addition,
of 45º C. The PCR products were verified by electrophoresis populations of A. brightwellii from Valerio Trujano and
in 1% agarose gel electrophoresis, purified by gel band Xochimilco, but not those of Zumpango, showed differences
purification kit (GE Healthcare) and cloning in pDrive in the cyst diameter (Fig. 2). Compared to inter-populational
(Qiagen) or pCR 2.1 TOPO (Invitrogen) vectors following differences, those of intra-strain are far greater. The smallest
the manufacturer’s instructions. The vectors were adults (about 700 µm) were from Valerio Trujano lake
sequenced in the DNA Sequencing Laboratory of our (Guerrero State) samples while the largest were from
university (UBIPRO, FES Iztacala), using the automated Xochimilco lake.The body size of A. brightwellii from
sequencer 3100 Genetic Analyzer (Aplied Biosystems). The Zumpango showed intermediate values. Similar tendencies
vectors were sequenced in both directions with the universal were reflected in trophi size and the diameter of resting
primers T7, SP6, M13 Reverse or M13 Forward. A total of eggs (Table 4).
650 and 789 bp was used for phylogenetic analysis with
CO1 gene and ITS region. Morphologically the cysts of A. brightwellii from
the three different waterbodies showed slightly different
The phylogenetic estimations among Asplanchna pattern (Fig. 2). The number of globular structures on the
brightwellii were done using PAUP 4.0b10 for 32.bit surface of cysts was smaller for Valeriano Trujillo strain,
Microsoft Windows (Sinauer Associates, Sunderland, USA), while these were larger and less numerous for both
and a heuristic search was carried out with 100 random Xochimilco and Zumpango lakes.
stepwise addition replicates. There was a maximum of 1000
trees for each replicate. Successive approximations were CO1 and ITS phylogenetic analysis : To determine if the
performed on data sets weighted a posteriori based on the CO1 and ITS sequence reflect the morphological and
rescaled consistency index derived from trees obtained from morphometric differences observed in the three clones of
A. brightwellii;we amplified these markers and obtained a
unweighted parsimony analyses. A total of 1000 replicates
650 bp of the CO1 gene and a 789 bp of ITS region. A total
bootstrap was done, and a consensus tree was obtained
of 13 sequences was aligned by CLUSTAL W program and
using the majority rule. The branches with less than 50%
obtained an ITS and CO1 gene tree by a heuristic search
bootstrap support were collapsed in the consensus tree.
based on maximum parsimony with a bootstrap of 1000
The multiple alignments were done using the ClustalX
iterations, resulting in the final consensus tree (and the
Program with default settings (Thompson et al., 1997).
branches were collapsed using majority-rule). The ITS
Results and Discussion region tree displayed two groups Xochimilco and Valerio
Trujano-Zumpango (Fig. 3a); this analysis did not reflect
Morphology and Morphometry : Selected data on the the morphological grouping; on the contrary, the CO1 gene
physico-chemical variables showed that altitude and tree separated the organisms according to morphological
temperature varied, while the other two parameters were clusters and location (Xochimilco, Valerio Trujano (Guerrero)
nearly similar (Table 1). The trophi of A. brightwellii is and Zumpango lakes) (Fig. 3b). In the CO1 gene sequence
different from other congeneric species and can be easily one hundred ten sites were variable and fifty five were
distinguished, for example from A. sieboldii that possesses parsimony informative sites, whereas for the ITS sequence,

Table 1 : Sample collection sites, mean temperature, pH, K and the number of individuals used for morphometry

Collection site Coordinates Altitude (m) Mean temp.(ºC) pH K Ind.

Xochimilco, Mexico City 19°15.190’N and 99° 5.559’W 2240 21 8.4 0.7 42
Zumpango, State of Mexico 19° 46.991’N and 99° 8.377’W 2200 19 9.7 0.9 45
Valerio Trujano, Guerrero State 18°18.294’N and 99°27.595’W 800 26 7.4 0.2 45

Journal of Environmental Biology, November 2013

Morphological and molecular analysis of Asplanchna 1043

Table 2 : Results of one way-ANOVA performed on the body Table 3 : Results of one way-ANOVA performed on the body
size, width, lengths of rami, bulla, spine on the inner margin of size, width, lengths of rami, bulla, spine on the inner margin of
rami and cyst diameter of A. brightwellii obtained from three rami and cyst diameter of three cultured populations of A.
freshwater bodies (Xochimilco lake, Zumpango lake and Valerio brightwellii obtained from Xochimilco lake, Zumpango lake or
Trujano lake) Valerio Trujano lake

Source of variation df SS MS F Source of variation df SS MS F

Body length 2 2169308 1084654 60.09*** Valerio Trujano population
Error 129 2328546 18050 Body length 2 141457 70728 5.72**
Body width 2 479220 239610 55.65*** Error 42 519573 12370
Error 129 555419 4305 Width 2 27537 13768 5.42**
Error 42 106720 2540
Rami 2 6275 3137 62.82***
Rami 2 75.80 37.90 1.55 ns
Error 129 6443 49.94 Error 42 1024 24.38
Bulla 2 599 299 29.82*** Bulla 2 34.45 17.22 3.01 ns
Error 129 1295 10.04 Error 42 240.18 5.71
Spine length 2 5.85 2.92 6.65** Spine length 2 1.11 0.55 2.33 ns
Error 129 56.76 0.44 Error 42 9.98 0.23
Resting egg diameter 2 3556 1778 27.16*** Resting egg diameter 2 683 341 5.39**
Error 42 2658 63
Error 132 8642 65.47
Xochimilco population
df = degrees of freedom, SS = sum of squares, MS = mean squares, Body length 2 20357 10178 0.35 ns
F = F-ratio, *** = p<0.001; ** = p<0.01 Error 39 1109700 28453
Width 2 9440 4720 0.94 ns
one hundred twenty two sites were variable and ninety six Error 39 196693 5043
sites corresponded to parsimony. Rami 2 4.36 2.18 0.02 ns
Error 39 3834 98.32
When the tree was built using the combination of Bulla 2 2.41 1.20 0.06 ns
both ITS and CO1 sequences (Fig. 4), the phylogenetic Error 39 725.84 18.61
relationships observed on CO1 gene were consistent, but Spine length 2 0.61 0.30 0.42 ns
showed differences with the those observed on ITS regions Error 39 28.23 0.72
tree (only two groups), taking into account that the DNA Resting egg diameter 2 722 361 6.59**
Error 42 2299 55
amplifications came from the same samples, the differences
Zumpango population
between ITS region and CO1 gene must probably only Length 2 15057 7528 0.60 ns
indicate a differential rate of divergence for the two Error 42 522400 12438
sequences. When the trees were re-done with the CO 1 Width 2 31864 15932 3.653*
gene sequence of Asplanchna sp. and the ITS region of Error 42 183163 4361
Asplanchna sieboldii the clustering were the same as CO1 Rami 2 142.67 71.33 2.20 ns
gene tree (Fig. 4) also the concatenated tree was consistent Error 42 1361.22 32.41
with the morphological classification. Bulla 2 10.31 5.16 0.767 ns
Error 42 282.58 6.73
Our study confirms that within a narrow Spine length 2 0.97 0.49 1.29 ns
geographical area (by about 500 km), there is considerable Error 42 15.86 0.38
difference in the morphology and morphometry of A. Resting egg diameter 2 124 62.13 1.21 ns
Error 42 2153 51.28
brightwellii. Samples from Valeriano Trujillo lake represent
typical tropical conditions while those from Zumpango and df =degrees of freedom, SS=sum of squares, MS=mean squares,
Xochimilco are from high altitude (Table 1). Depending on F=F-ratio, **=p<0.01, *=p<0.05; ns=non-significant (p>0.05)
the collection site, the morphology and morphometry of A.
brightwellii vary. For example, the morphological variations
less than 4% dry weight, this is not a preferred prey item to
have been reported within a given species of Asplanchna
the larval fishes (Wallace et al., 2006; Dumont, 2007).
(José de Paggi, 2002).
However, when Asplanchna feeds on other zooplankton,
Information about zooplankton predators its stomach appears coloured and in such cases fish larvae
(vertebrates and/or invertebrates) in the freshwater bodies easily locate and feed on such individuals (M. Gophen,
is helpful to understand the population abundances and personal communication). We have not analyzed the gut
adaptive strategies of A. brightwellii in a given waterbody. contents of larval fishes from the waterbodies from which
Since Asplanchna is a large transparent predator and with A. brightwellii was isolated. Certain predatory copepods

Journal of Environmental Biology, November 2013

1044 J.J. Contreras et al.

Fig. 2 : Morphology of cysts of cultured A. brightwellii. X = Xochimilco (Mexico City), VT: Valerio Trujano ( State of Guerrero); Z:
Zumpango (State of Mexico)

(Cyclopoidea) feed on Asplanchna (Brandl, 2005). Although et al., 2010). Therefore, ITS is thought to be alternative
we encountered cyclopoids in our plankton collections, approach for evaluating such species. The variability in
these were not quantified. genes of any species of Asplanchna is not well established.
However, it is largely believed that Asplanchna with shortest
Information about food resources of A. brightwellii generation time among all rotifer species (Dumont and Sarma,
from the chosen waterbodies is also scanty. We found 1995) is considered to one factor that could contribute to
Brachionus havanaensis, Keratella cochlearis and high amount of variation in genes. In addition, members of
Polyarthra vulgaris in relatively higher abundances (50 Asplanchna reproduce rapidly and build up populations
ind. l-1) in the zooplankton samples. Both Brachionus and that quickly crash with the production of cysts (Wallace
Keratella are preferred prey items for Asplanchna while and Snell,2010). The genetic recombination due to sexual
Polyarthra generally escapes predation (Chang et al., 2010). reproduction together with variable food and feeding habits
contribute to variations in the morphology of trophi and
The differences in the morphological traits are also
cysts.
reflected in the molecular analysis adapted in this work.
Both ITS and CO1 indicated results depending on the power From the maximum parsimony trees, it is evident that
of resolution of the techniques. It is generally assumed that populations of A. brightwellii from Valerio Trujano and
CO1 is a less powerful tool to evidence the genetic Zumpango were closely related than the either to
divergence among populations of the same species (Chen Xochimilco. The phylogenetic significance of this remains

Table 4 : Morphometric data on the body size, trophi size and cyst diameter of A. brightwellii cultured under laboratory conditions. For
each site, the measurements (µm) were based on three clones

Collection site Body size Trophi size (length) Resting egg

Length Width Rami Bulla Spine diameter
Valerio Trujano
692±28a,c 313±13a,c 77±1a,d 24±0.6a,d 3.6±0.1a,d 138±2a,c
783±36a,f 351±14a 76±1a 23±0.4a,e 3.8±0.1a,e 148±1 b,i
648±18b,c 291±11 b,c 74±2a 21±0.7a 3.4±0.1a,f 142±2 b,c
Xochimilco
1038±41d 480±21d 92±3 b 28±1.0b 3.4±0.2b,d 159±2 d,g
993±47d 447±16d 93±3 b 28±1.0b 3.6±0.2b,e 150±2e,i
1040±44d 476±20d 92±2b,e 28±1.0b 3.7±0.2b,f 157±2f,g,j
Zumpango
817±33e 357±26e 82±1 c,d 24±0.5c,d 3.2±0.1c,d 153±2h
847±28e, f 417±11f,h 83±1c 24±0.4c,e 3.0±0.1c,e 149±2h,i
861±25e 408±8 g,h 86±2c,e 25±1.0c 3.3±0.2c 152±2h,j
Shown are the mean ± SD based on 40-45 individuals. For each variable, data carrying similar alphabets are not significant (p>0.05)

Journal of Environmental Biology, November 2013

Morphological and molecular analysis of Asplanchna 1045

A. brightwellii Guerrero A. brightwellii Guerrero

(A) 99
A. brightwellii Guerrero (B) A. brightwellii Guerrero
63
100
A. brightwellii Guerrero A. brightwellii Zumpango
100 97
A. brightwellii Zumpango A. brightwellii Zumpango

A. brightwellii Zumpango A. brightwellii Xochimilco

A. brightwellii Xochimilco
A. brightwellii Xochimilco 100
100
A. brightwellii Xochimilco
A. brightwellii Xochimilco
Asplanchna sieboldii
Asplanchna sieboldii
Fig. 3 : Maximum parsimony trees of ITS region (A) and CO1 gene (B) resulting from analysis of the ITS region and CO1 gene sequences of
A. brightwellii species. The source of clone is indicated after the species name. Asplanchna sieboldii and Asplanchna sp. were used as outgroup.
Bootstrap values for 1000 replicates are shown above branches.

unclear at this stage. However, morphological analysis A. brightwellii Guerrero

showed that indeed the largest individuals of A. brightwellii
100
were observed from Xochimilco. The range of variation in A. brightwellii Guerrero
the body length and width as well as the morphology of
resting eggs of all the strains of A. brightwellii agree with 100
A. brightwellii Zumpango
the descriptions available in literature (José de Paggi, 2002). 98
The morphology of trophi, the size and nature of spine on A. brightwellii Zumpango
the inner margin of rami observed here also agree with the
earlier studies (Wallace et al., 2006; Wallace and Snell, 2010).
A. brightwellii Xochimilco
The resting egg morphology in rotifers is believed 100
to be species-specific (Wallace and Snell, 2010). In our study A. brightwellii Xochimilco
we found that the pattern of lobular surface of resting eggs
of A. brightwellii varied among the populations, although A splanchna sp.
the basic structure was similar to the description available
Fig. 4 : Maximum parsimony tree of combination of ITS region and
in literature (Wallace and Snell, 2010). Resting egg CO1 gene sequences of A. brightwellii. The source of clone is indicated
morphology, however, showed that strains from Xochimilco after the species name. Asplanchna sp. was used as outgroup.
and Zumpango more closely related than those of Guerrero.
Geographically, Xochimilco and Zumpango are closer (about
successfully applied to the rotifer Brachionus plicatilis
100 km) while Guerrero is wide apart (about 350 km).
complex (Gómez et al., 2002).
The results of the analysis of ITS region tree yielded
Many morphological traits including the body size
two groups of Xochimilco and Guerrero-Zumpango
of rotifers are inducible. For example, larger bodied
populations. However, this analysis did not reflect the
morphological grouping where there were indeed significant individuals of Brachionus species due to the presence of
differences among the strains of A. brightwellii with respect infochemicals from Asplanchna has been well documented
to the body size (length and width), length of rami, bulla (Gilbert, 1998). Depending on the prey availability, some
and spine on the inner margin of rami as well as the diameter species of Asplanchna change from smaller sized o large-
of resting eggs. On the contrary the CO1 gene tree separated bodied individuals (Wallace and Snell, 2010). Therefore field
the populations of A. brightwellii according to collected samples may not be at times fully appropriate for
morphological clusters and location (Xochimilco, Guerrero interpreting molecular data with morphological adaptations.
and Zumpango lakes). For certain taxa such as cnidarians, On the other hand, it is also possible that widespread
CO1 analysis is largely ineffective (McFadden et al.,2011). physiological and behavioral adaptations together with
Therefore other molecular techniques are adapted. As sexual communication through chemical signals may be
indicated earlier, ITS is useful for the species with variable responsible for the lack of morphological changes in certain
structure of mitochondrial genomes. ITS has been species of rotifers (Gómez et al., 2002).Therefore, a

Journal of Environmental Biology, November 2013

1046 J.J. Contreras et al.

combination of morphological traits and molecular approach of Asplanchna girodi (Rotifera) as a function of prey
(Anuraeopsis fissa) density. Hydrobiol., 306, 97-107 (1995).
would help resolve some interrelationships among species
Fontaneto, D., W.H. De Smet and G. Melone: Identification key to
or even clones (Birky et al., 2011). In our study we cultured the genera of marine rotifers worldwide. Meiofauna Marina,
all the strains of A. brightwellii under identical test 16, 75–99 (2008).
conditions including the quantity and the type of prey items. Gómez, A., M. Serra, G.R. Carvalho and D.H. Lunt: Speciation in
ancient cryptic species complexes: evidence from the molecular
In spite of this, we observed significant differences among
phylogeny of Brachionus plicatilis (Rotifera). Evolution, 56,
the strains, with reference to morphology and molecular 1431-1444 (2002).
analysis. It appears that physical barrier, geographical Hoffmann, A.A. and L.H. Rieseberg: Revisiting the impact of
distance, temperature, geological factors as well as the inversions in evolution: From population genetic markers to
drivers of adaptive shifts and speciation? Ann. Rev. Ecol. Evolu.,
presence of predators and prey items are probably
System., 39, 21 42 (2008).
responsible for the observed differences.These differences José de Paggi, S.: Rotifera, Vol. 6. Family Asplanchnidae. In: Guides
clearly show the need to consider strains of much wider to the Identification of the Microinvertebrates of the
geographical regions.Thus, in conclusion our morphological Continental Waters of the World. Backhuys Publishers (Eds.
T. Nogrady and H. Segers), The Hague, pp. 1–27 (2002).
and morphometric evaluations together with CO1 and ITS
McFadden, C.S., Y. Benayahu, E. Pante, J.N. Thoma, P.A. Nevarez
strongly indicate the existence of considerable variations and S.C. France: Limitations of mitochondrial gene barcoding
in the strains of A. brightwellii obtained from Central in Octocorallia. Mol. Ecol. Res., 11, 19-31 (2011).
Mexico. Nandini, S. and S.S.S. Sarma: Effect of starvation time on the prey
capture behaviour, functional response and population growth
Acknowledgments of Asplanchna sieboldi (Rotifera). Freshwater Biology, 42,
121-130 (1999).
Nandini, S., S.S.S. Sarma, R.J. Amador López and S. Bolaños Muñoz:
We thank two anonymous reviewers for constructive
Population growth and body size in five rotifer species in
comments on our manuscript. This study is supported by response to variable food concentration. J. Freshwater Ecology,
PAPCA (2010-1011-25 project). Partial financial assistance 22, 1-10 (2007).
was also obtained from PAPIIT-IN221111. JJC has received Ortells, R., A. Gómez and M. Serra: Effects of duration of the
planktonic phase on rotifer genetic diversity. Arch. Hydrobiol.,
doctoral scholarship (59114) from CONACyT (Mexico).
167, 203-216 (2006).
Schröder, T. and E.J. Walsh: Genetic differentiation, behavioral
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