Research Article |
Corresponding author: Gerardo Guerrero-Jiménez ( ggj_2049@hotmail.com ) Academic editor: Pavel Stoev
© 2019 Gerardo Guerrero-Jiménez, Patrizia Elena Vannucchi, Marcelo Silva-Briano, Araceli Adabache-Ortiz, Roberto Rico-Martínez, David Roberts, Roy Neilson, Manuel Elías-Gutiérrez.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Guerrero-Jiménez G, Vannucchi PE, Silva-Briano M, Adabache-Ortiz A, Rico-Martínez R, Roberts D, Neilson R, Elías-Gutiérrez M (2019) Brachionus paranguensis sp. nov. (Rotifera, Monogononta), a member of the L group of the Brachionus plicatilis complex. ZooKeys 880: 1-23. https://doi.org/10.3897/zookeys.880.28992
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The Brachionus plicatilis complex represents the most studied group of rotifers, although the systematics of the species complex has not been completely clarified. Many studies have been conducted trying to explore the diversity within the complex, leading to the recognition of three major morphotypes: large (L), small-medium (SM), and small (SS). Currently six species have been described and classified under these types and another nine taxa have been identified but not formally described. Within the L group, three species have been officially described [B. plicatilis s.s. (L1), B. manjavacas (L2), and B. asplanchnoidis (L3)], while a formal description of L4, unofficially known as B. ‘Nevada’, is still lacking. In the present study, a new species, Brachionus paranguensis sp. nov., is formally described and presented as a representative of the L4 clade. The species has been named after a high altitude saline crater lake from Central Mexico, where the specimens were collected. An integrated approach using DNA taxonomy through COI and ITS1 markers, morphology, and ecology was used to confirm the identity of the new species.
COI gene, ecology, ITS1, morphometry, rotifers, taxonomy.
The presence of multiple cryptic species that have been classified as a single species due to their morphological similarity still represents a major challenge for biologists (
In rotifers high levels of cryptic speciation occur (
1. B. asplanchnoidis Charin, 1947 (L)
2. B. plicatilis sensu stricto Müller, 1786 (L)
3. B. manjavacas Fontaneto et al., 2007 (L)
4. B. ibericus Ciros-Pérez et al., 2001 (SM)
5. B. koreanus Hwang et al., 2013 (SM)
6. B. rotundiformis Tschugunoff, 1921 (SS)
However,
In the present work, Brachionus paranguensis sp. nov., collected in the hypersaline and highly alkaline volcanic maar Rincón de Parangueo, Guanajuato, Mexico (
To support the identity of the new species an integrated approach using DNA taxonomy, ecology and morphology was applied. The DNA taxonomy was based on two genes, the mitochondrial gene COI (cytochrome c oxidase I), sometimes referred to as the DNA barcoding gene, and the nuclear ribosomal ITS1 (internal transcribed spacer I). Sequences of B. paranguensis sp. nov. were compared with published sequences belonging to the L group of the B. plicatilis species complex and phylogenetic analyses were performed to infer the relationship among sequences.
In order to provide further evidence to support the recognition of B. paranguensis sp. nov., a morphological description of parthenogenetic females and resting eggs is provided, as well as lifespan analysis. Furthermore, a formal description of the species is given along with potential diagnostic features specific to B. paranguensis sp. nov. that could discriminate this species from other members of the L group.
Water samples were collected with a zooplankton net of 50 μm from the three water bodies located in the Rincón de Parangueo volcano, Guanajuato, Mexico (WGS84 coordinates 20°25'46"N; 101°14'48"W, altitude 1686 m above the sea level). Samples used for taxonomic identification were fixed with 4% formalin, while those intended for DNA analysis were fixed with 96% ethanol. An additional in vivo sample was kept for culturing under laboratory conditions. This latter sample was kept in a cooler at 4 °C for no more than 4 hours until cultures were set up in the laboratory.
Physical and chemical parameters were measured in situ using the Yellow Spring Instruments Model 556 MPS probe. Environmental variables included: pH, temperature, dissolved oxygen (DO), conductivity and salinity [calculated with the conductivity to salinity conversion table by
From field samples ten specimens were selected for DNA analysis. DNA was extracted from each individual using a mixture of proteinase K with lysis buffer for invertebrates and digested overnight at 56 °C. Genomic DNA was subsequently extracted using the glass fiber membrane method in 2 μm Pall plates (
For ITS1 the PCR reactions were prepared using 8.85 μl of deionized water, 1.5 μl of 10X Buffer, 1 μl of BSA, 0.6 of μl MgCl2 (50mM), 0.3 μl of both primers (20 μM), 0.3 μl of dNTPs (10 mM) and 0.15 μl Taq polymerase (Platinum). The ITS1 primers used were III (5’-CACACCGCCCGTCGCTACTACCGATTG-3’) and VIII (5’-GTGCGTTCGAAGTGTCGATGATCAA-3’) of
All amplified products were screened with an agarose E-gel (Invitrogen) and pictures of the positives were taken. The products were labeled with the BigDye Terminator v. 3.1 (Applied Biosystems, Inc.) and sequenced bidirectionally. The DNA sequencing process was conducted at the James Hutton Institute of Dundee (Scotland, UK). All sequences were quality checked with Geneious 4.8 (http://www.geneious.com,
Both COI and ITS1 sequences of B. paranguensis sp. nov. were aligned against all B. plicatilis complex L clade COI and ITS1 sequences available in the NCBI (National Center for Biotechnology Information) public database. Sequences were trimmed to a sequence length of 550 bp for COI and to 350 bp for ITS1, and those that did not cluster with the members of the L group were discarded. A total of 665 COI and 148 ITS1 sequences belonging to the whole L group of the B. plicatilis complex were retained for this study. One COI sequence, KU299077 (
Sequence alignments were reduced to haplotypes by collapsing all identical sequences, using the online toolbox users-birc.au.dk/biopv/php/fabox/dnacollapser.php# (
A Light Microscope (LM) (Nikon Eclipse Ni) and a Scanning Electron Microscope (SEM) were used to analyze the morphology of B. paranguensis sp. nov. females, males, trophi, and eggs. Specifically, the presence of gastric glands, the shape of the dorsal sinus and the anterior spines of the female were analyzed by LM. Whereas, the lorica surface structure, the presence of lateral antennae, the foot aperture in the female, the male, the three different type of eggs, and the trophi, were analyzed by SEM. All samples were taken directly from the same field samples as the material for DNA analyses with the exception of the males, which were taken from laboratory cultures.
One hundred females, fifty males, twenty parthenogenetic eggs, twenty mictic eggs and twenty unfertilized eggs were extracted from the field samples and fixed in 4% formalin. Specimens were dehydrated using a graded ethanol series, starting with 60% and finishing with 100% ethanol; after which critical drying point was performed. Samples were then mounted in a SEM stub (1 cm high x 1.2 cm in diameter) and covered with gold. For the preparation of trophi a sample of 20 female organisms was used. Trophi were removed according to the methodology of
New-born females were isolated from cultures and placed in a well of a 24-well polystyrene plate for 48 hours. In order to compare morphometric data of B. paranguensis sp. nov. against the other members of the L group, females were fixed with 4 % formaldehyde and placed all in the same position under the optical microscope where twenty digital pictures were taken. Organisms were placed and measured according to
Culture conditions
Cultures were set up from several wild individuals collected in the volcanic maar Rincón de Parangueo, using the same salinity recorded in situ, 25 g L-1. Cultures were maintained in a bioclimatic chamber with a photoperiod of 16: 8 light: dark, a temperature of 25 °C and a medium with 25 g L-1 of RED-SEA SALT (Ca 410 ppm, Mg 1230 ppm, and AlK / KH Meq / l / 7.7 dKH) dissolved in distilled water (pH = 8.5). Rotifers were fed with 106 cells/ml concentration of the algae Nannochloropsis oculata.
Lifespan of parthenogenetic female and males
Lifespan analysis of males and females was conducted under laboratory conditions in order to provide ecological information of the species. In 1 mL of medium, 10 neonates of parthenogenetic females were isolated in individual wells of a 24-well polystyrene plate until they died. Every 24 hours neonates of each female were collected from the wells. Rotifers were fed with 106 cells/ml of Nannochloropsis oculata and placed into a new well with fresh medium in a total volume of 1 ml, every day during the experiment. The mean lifespan, the number of eggs per rotifer, and the maximum intrinsic growth rate were calculated for parthenogenetic females according to
From the 10 processed individuals of Brachionus paranguensis sp. nov., eight sequences were obtained for each marker, COI and ITS1, from the same individuals. For both markers, sequences belonged to a single haplotype (MK434153 and MH708047, respectively). The COI alignment that included 665 sequences belonging to members of the L group, collapsed into 146 haplotypes; while ITS1 alignment contained 148 sequences and collapsed into 12 haplotypes. The ML trees for both COI and ITS1, formed four well-defined clusters representing the four L clades of the B. plicatilis complex (
Comparison of COI and ITS1 LM trees focusing on the L4 group. COI and ITS1 Maximum Likelihood trees representing the four L groups of the Brachionus plicatilis complex and one outgroup species Brachionus rotundiformis. Focus is placed upon B. paranguensis sp. nov. L4 clade (grey shading) where details of the origin of each haplotype are given; all the other species are collapsed. Dotted lines between haplotypes indicate COI and ITS1 sequences that were sourced from the same individuals. Numbers at nodes represent support values (bootstrap = 1000). A list of references for each haplotype within the L4 group is provided.
COI genetic distances among and within the members of the L group. COI uncorrected p-distances generated within and among members of the L group. In black are within distances, while lower left are between mean p-distances.
L1 | L2 | L3 | L4 | |
---|---|---|---|---|
L1 | 3.6% | |||
L2 | 19.8% | 2.8% | ||
L3 | 18% | 19.7% | 4% | |
L4 | 18.9% | 17.7% | 15% | 8.5% |
ITS1 genetic distances among and within the members of the L group. ITS1 uncorrected p-distances generated within and among members of the L group. In black are within distances, while lower left are between mean p-distances. Within distance for L2 has N/A as only one haplotype is present.
L1 | L2 | L3 | L4 | |
---|---|---|---|---|
L1 | 0.7% | |||
L2 | 8.9% | N/A | ||
L3 | 6.3% | 9.3% | 0.6% | |
L4 | 2.8% | 7.6% | 5% | 0.001% |
Superorder Pseudotrocha Kutikova, 1970
Order Ploima Hudson & Gosse, 1886
Family Brachionidae Ehrenberg, 1838
The volcano Rincón de Parangueo, Guanajuato, Mexico, has two or three water bodies inside the crater, depending on the season, and B. paranguensis sp. nov. was present in one of them, 20°25'46"N; 101°14'48"W, at the altitude of 1686 m above sea level (Fig.
In order to confirm the identity of B. paranguensis sp. nov., hundreds of individuals from field and culture samples representing all stages (amictic females, males, resting eggs, parthenogenetic eggs, and unfertilized sexual eggs), and around 30 trophi, were examined by LM and SEM. Specifically, hundreds of females were used to take morphometric measurements, and dozens of males were observed to analyze their morphological features. Moreover, diapausing eggs, parthenogenetic eggs and unfertilized eggs were examined and pictures of each egg were taken. Finally, approximately 30 trophi were analyzed in order to compare their morphological features with those of the other eggs belonging to the L group.
Holotype. A parthenogenetic female mounted on a slide with a mix of formaldehyde-glycerol sealed with DePex medium, deposited in the Zooplankton Reference Collection of El Colegio de la Frontera Sur with accession number ECO-CH-Z-10331. Paratypes. Two slides with a parthenogenetic female, deposited in the Zooplankton Reference Collection of El Colegio de la Frontera Sur with accession numbers ECO-CH-Z-10332, 10333.
Parthenogenetic female : No clear morphological differences were observed between B. paranguensis sp. nov. and the other species belonging to the L group of the B. plicatilis complex with respect to the anterior dorsal spine, the U-shape sinus, the orange peel like surface of the lorica, and the presence of gastric glands. An exception is B. asplanchnoidis, whose lorica presents an elongated and wider shape, and the antero-ventral U-shape sinus is wider compared to that of the other members.
Trophi: In B. paranguensis sp. nov. satellites are robust and there are sharp projections in the inner upper margin; basifenestras with similar size and shape, ramus with two posterior projections, the left one smaller and thinner than the right one. In B. plicatilis s.s. the shape of the satellites is triangular with no projection; ramus with two posterior projections, the left one bigger and wider than the right one; basifenestras asymmetrical with different sizes. In B. manjavacas the shape of the satellites is triangular and sharper compared to B. plicatilis s.s.; ramus with two posterior projections, the left one bigger and wider than the right one; basifenestras with same shape but different size, the left one bigger than the right one. In B. asplanchnoidis satellites are robust and the projections in the inner upper margin is rounded; ramus with two posterior projections, the left one smaller and thinner than the right one, the right one with cylindrical shape; basifenestras with different shape and size, the left one smaller than the right one.
Resting eggs: Oval shape in B. paranguensis sp. nov., B. plicatilis s.s. and B. manjavacas, although the two latter species also present small holes on the surface of the eggs (
Ecology: B. paranguensis sp. nov. grows preferentially in salinities higher than 25 g L-1. B. plicatilis s.s. grows better in salinities from 5 to 15 g L-1 (
The population of B. paranguensis sp. nov. from Rincón de Parangueo volcano was used to formally describe all individuals belonging to the L4 group of the B. plicatilis complex. Parthenogenetic females (Fig.
Male
(Fig.
Taxonomic features of B. paranguensis sp. nov. A parthenogenetic female with lateral antennae (LA), gastric glands (gg), an U-shape sinus (us) B anterior dorsal and ventral spines taken by LM and SEM C lorica with an orange peel like surface D foot aperture E lateral antenna (see arrow) F SEM microphotographs of the female, ventral plate and G dorsal plate and H male. Scale bars: 50 µm (A, B, D, F, G, H), 10 µm (C, E). All females from sample collected in June 27, 2015. Males from cultures obtained from females collected in the volcanic maar Rincón de Parangueo.
Measurements of all species belonging to the L group. Measurements of adult females between all species belonging to the L group, according to
Group L | Measurements | ||||||||
a | b | c | d | e | f | g | h | i | |
B. plicatilis s. s. (L1) | 283.63 ± 6.38 | 122. 36 ± 1.72 | 203.62 ± 5.49 | 27.33 ± 0.42 | 13.74 ± 0.31 | 132.44 ± 2.13 | |||
B. manjavacas (L2) | 256.28 ± 3.9 | 109.37 ± 1.75 | 177.67 ± 2.62 | 21.74 ± 0.52 | 10.04 ± 0.28 | 125.09 ± 1.91 | |||
B. asplanchnoidis (L3) | 295.12 ± 8.1 | 126.12 ± 1.85 | 204.58 ± 4.8 | 26.64 ± 0.75 | 14.42 ± 0.45 | 130.88 ± 2.12 | |||
B. paranguensis sp. nov. (L4) N = 20 | 216.97 ± 13.78 | 106.43 ± 8.13 | 159.93 ± 10.93 | 25.43 ± 2.88 | 23.82 ± 2.23 | 20.13 ± 2.06 | 9.75 ± 0.81 | 12.93 ± 1.09 | 126.21 ± 7.72 |
Egg types: The outer membrane of the resting eggs presented a slightly rough ornamentation (Fig.
Trophi: Malleate type with all the characters of the genus (Fig.
Types of eggs in B. paranguensis sp. nov. SEM micrographs of the different types of eggs of B. paranguensis sp. nov. A resting egg from field samples and its special ornamentation and zoom of its membrane, see arrows (A.a, A.b) B parthenogenetic egg and C unfertilized sexual egg, both from cultured samples. Scale bars: 20 µm
Trophi components of B. paranguensis sp. nov. SEM pictures of the trophi components of B. paranguensis sp. nov., collected in June 27, 2015 A ventral view B dorsal view C rami and fulcrum D manubrium E satellites F unco and sub uncus ar: articulation of manubrium, m: membrane; ma: manubrium with posterior claw, see arrow (D); mmc: manubrium middle crest, see arrow (D); rr: reinforced ridge; st: satellites, see arrow (E); su: sub uncus; u: uncus.: b: basifenestras, see arrow (C); f: fulcrum, see arrow (C); mc: manubrium cavities; ra: rami, and projections of the posterior portion of the rami, see arrow (C)). Scale bars: 10 µm.
The specific name refers to the type locality, the volcanic maar Rincón de Parangueo.
According to the DNA sequences available in NCBI, B. paranguensis sp. nov. individuals were reported in Mexico, USA, Chile, Europe, Australia, and Japan. The habitat of B. paranguensis n. sp is represented by high salinity environments (>25 g L-1). Physical and chemical parameters of the maar where specimens were collected: Temperature= 28 °C; Conductivity= 43.2 mS/cm-1; Dissolved Oxygen = 5.5 mg L-1; pH= 11.07.
Under laboratory conditions (25 °C, 25 g L-1 salinity, 106 cells/ml of Nannochloropsis oculata provided as food) amictic females had a maximum lifespan of 10.8 days. Eggs per rotifer were 15.4 ± SD 5.8, and the maximum intrinsic growth rate was 0.49 (N = 10).
Due to the morphological stasis of the external characters that characterizes the B. plicatilis species complex (
The current taxonomy of the L group has not been resolved yet, leading to an underestimation of the true diversity present within the complex (
Phylogenetic analyses using both markers clustered B. paranguensis sp. nov. and B. ‘Nevada’ together as a single species, although a higher genetic variability in COI was observed within haplotypes (8.5%, ranging from 0 to 15%). Sequences of B. paranguensis sp. nov. revealed no variability in ITS1 with three clones of B. ‘Nevada’ available in GenBank, representing therefore a single haplotype, with only a single base-pair difference with the other haplotype belonging to a clone of B. plicatilis collected from Chile, included in the L4 clade.
Previous genetic analysis with the COI gene reported high genetic divergences within the B. plicatilis species complex up to about 20% (
Morphological differentiation among parthenogenetic females belonging to the L group species of the B. plicatilis complex is poor. The high variability in size and the lack of differentiation between the anterior dorsal and ventral margins, already reported for B. asplanchnoidis by
Trophi are indeed relatively consistent features in rotifers (
The study of resting eggs morphology could represent another alternative for species differentiation. Indeed, in rotifers, evidence of species specificity in resting eggs was already documented by
Differences in salinity preference among the species belonging to the L group may represent another factor thet al.ows discriminating between species. Nevertheless, no studies have been carried out on specific salinity preferences of the L group species. Available data reveal that in B. plicatilis s.s, a peak in the population growth rate was obtained at a salinity of 10 to 15 g/L-1, and then declined when salinity increased (
A formal taxonomic description has been provided for B. paranguensis sp. nov. combining DNA taxonomy, morphology and ecology, and results confirmed the identity of the species within the L group.
Based on DNA taxonomy, both COI and ITS1 markers placed B. paranguensis sp. nov. within the L4 clade of the B. plicatilis complex, confirming that B. paranguensis sp. nov. and B. ‘Nevada’ represent the same taxonomic identity. Results of genetic variability within the L4 clade were different among markers, with a higher COI intraspecific variability (8.5%) compared to the low 0.001% divergence in the ITS1. High genetic divergence in the COI marker is not unusual and its reliability for predicting species in the B. plicatilis complex has already been questioned due to its high mutational rate that leads to an over-splitting of the species. ITS1 has been therefore suggested as a more reliable marker for DNA taxonomy.
Comparison of SEM images among the trophi of B. paranguensis sp. nov., B. plicatilis s.s., B. manjavacas, and B. asplanchnoidis, indicate clear differences, including basifenestras, ramus posterior projections and satellites. Resting eggs morphotypes seemed to be species specific, although a comparison with B. asplanchnoidis resting eggs is also necessary. A differentiation of parthenogenetic females is only possible between B. asplanchnoidis and the other members of the L group, as no clear diagnostic characters are observed between females of the other L clades.
Ecological results on salinity preferences showed that B. paranguensis sp. nov. is the species better adapted to hypersaline water bodies. Indeed, results demonstrated that high salinity (>25 g L-1) positively affected individuals of B. paranguensis sp. nov., while the other members of the L group presented an optimum growth rate at salinities lower than 15–16 g L-1.
This work is a contribution of the Mexican Barcode of Life Network (MEXBOL) supported by CONACYT (251085). This article is in memory of Gloria and Balthazar, the first author’s aunt and uncle. Authors appreciate the discussions with Paul Hebert, Teresa Crease and Sean Prosser from the University of Guelph to understand concerted evolution and discordance between ITS1 and COI genes.
Figures S1–S2
Data type: molecular data
Explanation note: Figure 1. COI (TN93+G) Maximum Likelihood tree obtained with PhyML 3.0 of 146 haplotypes belonging to the four L clades of Brachionus plicatilis complex and one outgroup species Brachionus rotundiformis (AF387287). Figure 2. ITS1 (GTR+G) Maximum Likelihood tree obtained with PhyML 3.0 of 12 haplotypes belonging to the four L clades of Brachionus plicatilis complex and one outgroup species Brachionus rotundiformis (AF387239).