http://orcid.org/0000-0001-5804-4805
Figures
Abstract
Microorganisms, including rotifers, are thought to be capable of long distance dispersal. Therefore, they should show little population genetic structure due to high gene flow. Nevertheless, substantial genetic structure has been reported among populations of many taxa. In rotifers, genetic studies have focused on planktonic taxa leaving sessile groups largely unexplored. Here, we used COI gene and ITS region sequences to study genetic structure and delimit cryptic species in two sessile species (Limnias melicerta [32 populations]; L. ceratophylli [21 populations]). Among populations, ITS region sequences were less variable as compared to those of the COI gene (ITS; L. melicerta: 0–3.1% and L. ceratophylli: 0–4.4%; COI; L. melicerta: 0–22.7% and L. ceratophylli: 0–21.7%). Moreover, L. melicerta and L. ceratophylli were not resolved in phylogenetic analyses based on ITS sequences. Thus, we used COI sequences for species delimitation. Bayesian Species Delimitation detected nine putative cryptic species within L. melicerta and four putative cryptic species for L. ceratophylli. The genetic distance in the COI gene was 0–15.4% within cryptic species of L. melicerta and 0.5–0.6% within cryptic species of L. ceratophylli. Among cryptic species, COI genetic distance ranged 8.1–21.9% for L. melicerta and 15.1–21.2% for L. ceratophylli. The correlation between geographic and genetic distance was weak or lacking; thus geographic isolation cannot be considered a strong driver of genetic variation. In addition, geometric morphometric analyses of trophi did not show significant variation among cryptic species. In this study we used a conservative approach for species delimitation, yet we were able to show that species diversity in these sessile rotifers is underestimated.
Citation: Kordbacheh A, Wallace RL, Walsh EJ (2018) Evidence supporting cryptic species within two sessile microinvertebrates, Limnias melicerta and L. ceratophylli (Rotifera, Gnesiotrocha). PLoS ONE 13(10): e0205203. https://doi.org/10.1371/journal.pone.0205203
Editor: Ulrike Gertrud Munderloh, University of Minnesota, UNITED STATES
Received: June 28, 2018; Accepted: September 20, 2018; Published: October 31, 2018
Copyright: © 2018 Kordbacheh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. DNA sequences are available in GenBank. Accession numbers are provided in S1 Table. Trophi images are provided in S2 Fig and at UTEP Bioinformatics Data Repository, http://datarepo.bioinformatics.utep.edu/getdata?acc=468Y2C8Q43EZY9T.
Funding: This research was supported by grants from National Science Foundation (DEB 1257068 EJW, 1257116 to RLW). Funds from these grants were used by the authors to conceive the study design, collect and analyze data, and prepare the manuscript for publication. https://www.nsf.gov/. National Institutes on Minority Health and Health Disparities (2G12MD007592) [a component of the National Institutes of Health (NIH)]. Funds from this grant were used by the authors for data collection and analysis. https://www.nimhd.nih.gov/. Sigma Xi Grants-in-Aid of Research (G2012162274 to AK). Funds from this grant were used for data collection. https://www.sigmaxi.org/programs/grants-in-aid. Dodson Research Grant from University of Texas at El Paso to AK (2014 and 2015). Funds from this grant were used for data collection. https://www.utep.edu/graduate/_Files/dodson-research-grantdetailsinstructions.pdf. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Microorganisms are capable of long distance dispersal thus it has been suggested that they have cosmopolitan distributions with little geographic structure [1–4]. However, biogeographical patterns have been documented for many taxa such as soil [5] and marine bacteria [6–8], protists [9,10], fungi [11], and rotifers [12,13]. One of the reasons that microorganisms are often considered ubiquitous is the failure to identify cryptic species [14,15]. However, cryptic species complexes commonly occur in these taxa (e.g., [16–19]).
Similar to the other microorganisms, rotifers have passive dispersal through their dormant stages [20,21]. Thus, researchers inferred that rotifers have cosmopolitan distributions (e.g., [22]), and thus should show little population genetic structure due to gene flow. However, incorporating molecular tools in the study of rotifer diversity has provided ample evidence of cryptic species complexes and substantial genetic structure within and among rotifer populations (e.g., [23–30]). By definition cryptic species are not readily distinguished by morphology, but subtle morphological variations have been detected among species in some complexes. For instance, size and shape of lorica varies among cryptic species of Brachionus plicatilis Müller, 1786 (e.g., [31–36]), trophi and resting egg morphology differ in the Epiphanes senta (Müller, 1773) complex [37], and trophi size varies among populations of Rotaria magnacalcarata (Parsons, 1892) [38].
Genetic differentiation and the rate of diversification may vary among aquatic taxa with different life histories [39–41] and/or habitat differences [42]. Rotifer species can be planktonic, associated with littoral vegetation, or sessile [20]. In littoral zones there is higher habitat diversity provided by vegetation, debris, mosses, and filamentous algae as compared to the pelagic zone [20]. Habitat preference has been reported for some rotifers inhabiting littoral zones such as Collotheca campanulata (Dobie, 1849) [43–44] and Euchlanis dilatata Ehrenberg, 1830 [44]. Difference in habitat preference may limit connectivity among populations and cause genetic divergence [45]. Therefore, as a result of habitat differentiation, substantial genetic diversity is expected within and among populations [46]. In addition, genetic divergence among populations may be related to dispersal capabilities. For example, Russo et al. [47] found that genetic variation in 16 allozyme markers within two anemone species, Bunodosoma caissarum Correa, 1987 and Actinia bermudensis McMurrich, 1889 was related to their dispersal ability. B. caissarum has a long planktonic larval period that provides high dispersal capability. This species showed lower genetic structure compared to that of A. bermudensis with low dispersal abilities. Similarly, Lee et al. [48] found high genetic divergence among populations of the jelly Rhizostoma octopus (Linnaeus 1788) with bipartite life history (ΦST ≤ 0.75) and Stopar et al. [49] found low genetic differentiation within the holoplanktonic jelly Pelagia noctiluca (Forskål 1775) (ΦST ≤ 0.09) in the COI gene. Genetic differentiation within these cnidarian species may be associated with the variation in their dispersal capabilities. Therefore, sessile rotifers may show higher genetic structure than planktonic groups because in sessile rotifers females are mobile only during their larval stage, which can limit their dispersal range. However, similar to non-sessile rotifers, their resting stage may be transported across long distances.
Almost all studies on genetic structure and cryptic species of monogonont rotifers focus on planktonic taxa, with little attention having been paid to sessile species. Sessile rotifers of orders Collothecaceae and Flosculariaceae from superorder Gnesiotrocha are common in a wide assortment of aquatic habitats and attach to varied substrata [50]. Study of these forms is challenging as their plant substrata must be inspected to find them and several diagnostic characteristics need to be examined in live individuals [20]. Thus, they are often overlooked, which has led to gaps in our knowledge about their taxonomic diversity. Molecular tools have been applied in few phylogenetic studies that have included sessile rotifers (e.g., [51,52]), but these have not focused on examination of population level genetic patterns or detection of cryptic species.
One poorly studied sessile taxon is the genus Limnias (Flosculariidae), in which six morphospecies are currently recognized [53,54]: Limnias ceratophylli Schrank, 1803; L. cornuella Rousselet, 1889; L. melicerta Weisse, 1848, L. myriophylli (Tatum, 1868), L. nymphaea Stenroos, 1898, and L. shiawasseensis Kellicott, 1888. Limnias melicerta and L. ceratophylli are considered cosmopolitan and each has been reported from seven biogeographical regions [43]. The other four species have restricted distributions. Limnias cornuella has only been reported from Palearctic [55], L. myriophylli is reported from Afrotropical and Palearctic [43], L. nymphaea from Palearctic, and L. shiawasseensis from Nearctic biogeographical regions [43]. The few available studies on this genus include the following: taxonomy [54–58], trophi descriptions [51,59], tube formation [58], tube ultrastructure of L. melicerta [60], post-natal development [61], phylogeny of Flosculariaceae [51], ecological studies (i.e., water quality and abundance (L. melicerta; [62,63]), population growth (L. melicerta and L. ceratophylli; [64]), and toxicology [65]). To our knowledge, there are no published studies on genetic population structure within or among species of this genus.
We hypothesize that sessile rotifers will have a discernable genetic structure that will be more pronounced than that of planktonic rotifers and that this structure is sufficient to define independently evolving lineages as cryptic species. To test these hypotheses we studied genetic structure, identified cryptic species, and investigated geographic isolation of genotypes in Limnias melicerta and L. ceratophylli. We used partial mitochondrial cytochrome c oxidase subunit I (COI) gene and the internal transcribed spacer (ITS) region sequences from a broad geographic range. Partial 18S rRNA sequences were used to confirm monophyly of the two species. In addition, we examined morphological variation in trophi among putative cryptic species within L. melicerta and L. ceratophylli.
Materials and methods
Sample collection and culture
Aquatic plant samples were collected from habitats across the USA and a sediment sample from Australia (S1 and S2 Tables). Limnias melicerta and L. ceratophylli were identified and isolated from rehydrated sediments or by removing a piece of vegetation to which they were attached. Species identification was based on tube structure, shape of corona, antennae length, and the number of dorsal nodules [55]. Clonal lineages initiated from single females were cultured in modified MBL media [66] and fed a mixture of the algae Chlorella vulgaris Berijerinck, 1890 (The UTEX Culture Collection of Algae at the University of Texas at Austin [UTEX] strain 30) and Chlamydomonas reinhardtii Dangeard, 1888 (UTEX strain 90). Rotifers in this genus produce tubes of hardened secretions [20]; we added powdered carmine (Alfa Aesar, UK) to lab cultures to provide a supplementary matrix to aid tube construction and to increase their visibility in culture.
Voucher specimens were deposited in the UTEP Biodiversity Collections at The University of Texas at El Paso (L. melicerta: UTEP:Zoo:43, 105–134; L. ceratophylli: UTEP:Zoo:32–42, UTEP:Zoo:52–61). Deposited specimens included approximately 10 individuals from each population preserved in 95% ethanol and 10 clonal individuals preserved in 4% buffered formalin for molecular analyses and identification, respectively.
DNA extraction and gene amplification
DNA was extracted from one individual of each clonal lineage by adding 13 μl Chelex-100 (Bio-Rad Laboratories, CA, USA) and incubating at 100°C for 10 min. DNA templates were stored at -80°C until used for amplification. Number of clonal lineages examined from each population is given in S1 and S2 Tables.
An approximate 630 bp portion of the cytochrome c oxidase subunit I (COI) gene was amplified using the primers LCO1490: 5' -GGTCAACAAATCATAAAGATATTGG-3' and HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' [67]. The entire nuclear internal transcribed spacer region (ITS) was amplified using the primers ITS4: 5'-TCCTCCGCTTATTGATATGC-3' and ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' [68], and 865 bp of the 18S rRNA gene was amplified using primers 3F: 5’-GTTCGATTCCGGAGAGGG-3’ as modified by Giribet et al. [69] and primer 18Sbi: 5’-CTAGAGTCTCGTTCGTTATCGG-3’ as modified by Whiting et al. [70].
PCR reactions contained 10 μl of genomic DNA, 1 μl of each primer (500 ng/ μl), 22 μl HPLC grade sterile water, 1 μl GoTaq G2 DNA Polymerase (Promega), 10 μl 5X PCR buffer B (10 mM MgCl2, pH 8.5, Invitrogen) or 5X PCR buffer A (7.5 mM MgCl2, pH 8.5, Invitrogen), followed by adding 5 μl dNTP mix (2.5 mM each of dATP, dCTP, dGTP, dTTP) at 80°C. PCR cycles were run on a thermocycler (Techne TC-412) and consisted of an initial denaturation at 94°C for 1 min, followed by denaturation at 94°C for 1 min, annealing at 48°C for 2 min and extension at 72°C for 3 min for 35 cycles, and a final extension step at 72°C for 7 min. To verify the size of amplification products we used electrophoresis, and we purified them using GENECLEAN kits (MP Biomedicals, LLC) before sequencing. Sequencing was done at UTEP’s BBRC Genomic Analysis Core Facility on an Applied Biosystems 3130xl Genetic Analyzer using BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems). GenBank accession numbers for all sequences obtained are given in S1 Table (L. melicerta) and S2 Table (L. ceratophylli). The COI gene sequences of L. melicerta (accession number, KT870155.1) and L. ceratophylli (KT870157.1) from GenBank are not included in our analyses for two reasons. 1) The COI sequence of L. melicerta KT870155.1 is 330 bp, which was much shorter than COI sequences obtained in this study (623 bp). 2) The COI sequence of L. ceratophylli KT870157.1 grouped with cryptic species M of L. melicerta in phylogenetic analyses. Additional 18S rRNA sequences (L. melicerta: KM873599.1, L. ceratophylli: KM873598.1) and a COI sequence from L. melicerta (KT870154.1) were included from GenBank. Sinantherina socialis (Linneaus, 1758) and Ptygura pilula (Cubitt, 1872) were included as outgroups in phylogenetic analyses for the COI gene. Floscularia conifera (Hudson, 1886) and Ptygura brachiata (Hudson, 1886) were used as outgroup taxa in phylogenetic analyses based on ITS region. For analysis of 18S rRNA sequences, we used Collotheca campanulata as the outgroup taxon (S1 Table).
Genetic diversity
FinchTV v 1.4.0 [71] was used to check sequences manually, especially for potential double peaks in the ITS region sequences. The ITS region alignment was uploaded to the SeqPhase online tool (http://seqphase.mpg.de/seqphase/) to phase the sequences as described by Flot [72]. Contigs for all sequences were made using CAP 3 [73] and were aligned using MAFFT v 7 [74]. Mesquite v 3.2 [75] was used to manually check the alignments and to translate COI gene sequences to proteins. To measure substitution saturation, we used DAMBE v 6 [76]. Number of polymorphic sites, number of parsimony informative sites, number of haplotypes, haplotype diversity (h), and nucleotide diversity (π) were calculated using DnaSp v 5.10.01 [77], and uncorrected pairwise sequences distances ("p") were calculated in Mega v 7.0 [78]. A haplotype network was constructed using the median joining method in Network v 5.0.3 [79].
Species delimitation
Models for sequence evolution were TPM2uF+I+G for the COI gene, TPM1uF+I for the ITS region, and JC for the 18S rRNA gene as determined using Jmodeltest2 [80,81] available at the CIPRES Science Gateway 3.3 [82]. To construct the phylogenetic trees, Bayesian analysis was run for 107 generations with two parallel runs and a 25% burn-in period using MrBayes v 3.2.6 on XSEDE high-throughput computing resources available at CIPRES Science Gateway [82]. Phylogenetic analyses were implemented in BEAST and *BEAST [83] using GTR+I+G model of sequence evolution for the COI gene and GTR+I model for the ITS region. TPM2uF and TPM1uF models are not available in BEAST. However, both of these models are classified under the GTR model. Thus GTR was used in both instances.
To determine the number of evolutionary entities (putative cryptic species), we used Generalized Mixed Yule Coalescent (GMYC, [84]), Poisson Tree Process (PTP, [85]), Automatic Barcoding Gap Discovery (ABGD, [86]), *BEAST v 1.8.3 [83], and Bayesian Species Delimitation (BSD) implemented in Bayesian Phylogenetics and Phylogeography software (BPP v 3.1, [87–89]).
We used BEAST v 1.8.3 [83] to construct ultrametric trees and *BEAST v 1.8.3 [83] for species delimitation. Both analyses were run for the COI gene and ITS region sequences separately for 107 generations, with sampling every 1,000 generations. Tracer v 1.6.0 [90] was used to check the effective sample size (ESS>200) and to verify convergence. Consensus trees were obtained using TreeAnnotator v 1.8.3 with a 25% burn-in. Ultrametric trees were used for species delimitation in single threshold and multiple threshold GMYC [91] (http://species.h-its.org/gmyc/, accessed June 12, 2018), Bayesian GMYC (bGMYC) [92] and PTP methods. bGMYC was run using the R package bGMYC v 1.0.2 for 100,000 iterations with sampling every 1,000 iterations. We ran PTP by uploading the ultrametric trees to the online tool available at http://species.h-its.org/ptp/ (accessed June 12, 2018) and used default settings. ABGD delimitation was done by uploading the sequence alignment to the online tool available at www.abi.snv.jussieu.fr/public/abgd/ under the default settings (accessed June 12, 2018).
*BEAST v 1.8.3 [83] was run under assumptions regarding the number of species for both the COI gene and ITS region sequences. Lineages having posterior probabilities > 0.90 were retained in *BEAST analyses. To run BSD, the phylogenetic tree based on Bayesian inference was used as the guide tree, and we used the joint species delimitation and tree estimation method (unguided species delimitation) that does not rely on the topology of the guide tree.
Isolation by distance
Geographic distance matrices were constructed using Geographic Distance Matrix Generator v 1.2.3 [93]. To test the correlation between genetic variation and geographic distances (log transformed; km) among populations, Mantel tests with 10,000 permutations were run using the R package ecodist v 1.2.9 [94].
Trophi morphology
Trophi were prepared for scanning electron microscopy (SEM) by dissolving rotifer tissue in ~5% sodium hypochlorite, rinsing with deionized water 10–15 times, and air-drying on circular cover slips at room temperature [95]. Trophi were coated with gold/palladium using a Gatan 682 PECS sputter coater. SEM images were obtained at 20 kV using a Hitachi S-4800 system. Trophi were prepared for individuals from one clonal lineage from each examined population.
We used a geometric morphometric approach to study variation in the shape and size of 92 trophi for L. melicerta and 60 trophi for L. ceratophylli among putative cryptic species. This method uses Cartesian coordinates for a set of anatomical landmarks [96]. SEM images were obtained from caudal and frontal views of the trophi. Using TPS series software [97], nine landmarks on the caudal view and 10 on the frontal view of the trophi were digitized (Fig 1). Configuration of landmarks were analyzed using Generalized Procrustes Analysis [96]. Trophi size was calculated as Centroid Size (CS): i.e., the square root of the sum of squared distances between landmarks and their centroid [98]. Variation in the shape of trophi based on landmarks was examined using Discriminant Analysis in SPSS v 24 [99]. Because trophi centroid size was not normally distributed, variation in size among putative cryptic species was tested using a non-parametric Kruskal-Wallis test, and between the two morphospecies using a non-parametric Mann-Whitney U test implemented in SPSS v 24 [99].
A: frontal view of Limnias ceratophylli trophi, 1, 2: midpoint of manubrium, 3, 4: base of first large tooth of ramus, 5, 6: base of second large tooth of ramus, 7–8: tip of first large tooth of ramus, 9, 10: tip of second large tooth of ramus. B: caudal view of L. ceratophylli trophi, 1, 2: apical point of manubrium, 3, 4: midpoint of manubrium, 5: apical point of fulcrum, 6, 7: apical point of ramus, 8, 9: basal point of ramus. C: frontal view of Limnias melicerta trophi. D: caudal view of L. melicerta trophi.
Methods used in this study are available at protocols.io, dx.doi.org/10.17504/protocols.io.tppemmn.
Results
Genetic diversity
For the COI gene, sequences were analyzed for 72 individuals from Limnias melicerta and 25 individuals from L. ceratophylli. The ITS region was sequenced for 76 individuals from L. melicerta and 35 individuals from L. ceratophylli. For 18S rRNA, sequences were acquired for 17 individuals of L. melicerta and 18 individuals of L. ceratophylli. Alignment length was 623 bp for the partial COI gene sequences, 763 bp for the ITS region including insertions, and 865 bp for partial 18S rRNA gene sequences. COI gene, ITS region, and 18S rRNA sequences were not saturated (index of substitution saturation < critical index of substitution saturation, p < 0.001, S3 Table). For the COI gene, haplotype diversity was 0.87 for L. melicerta and 0.94 for L. ceratophylli; nucleotide diversity was 0.13 for L. melicerta and 0.11 for L. ceratophylli. The overall genetic distance in the COI gene among populations was 0–22.7% for L. melicerta and 0–21.7% for L. ceratophylli, and the genetic distance between L. melicerta and L. ceratophylli ranged from 19.2 to 24.1%. For the ITS region, haplotype diversity was 0.67 for L. melicerta and 0.56 for L. ceratophylli, nucleotide diversity was 0.01 for L. melicerta and 0.02 for L. ceratophylli. The overall genetic distance in the ITS region among populations was 0–3.1% for L. melicerta and 0–4.4% for L. ceratophylli, and the genetic distance between L. melicerta and L. ceratophylli was 1.2–5.6%. We also found seven heterozygous individuals for the ITS region in L. melicerta and two in L. ceratophylli. Based on 18S rRNA sequences, haplotype diversity, nucleotide diversity, and genetic diversity were 0 for both L. melicerta and L. ceratophylli. The genetic distance (uncorrected “p” distance) between L. melicerta and L. ceratophylli was 0.5%. Genetic diversity measures for all markers are summarized in Table 1.
Species delimitation
In two of the phylogenetic trees (i.e., based on COI gene and 18S rRNA sequences), monophyly of L. melicerta and L. ceratophylli was supported (Fig 2 and S1 Fig). Using 18S rRNA sequences, there were only two highly supported clades, one with each species. Additionally, L. melicerta and L. ceratophylli co-occurred in Moon Lake, WI. Each clustered with their conspecifics from other habitats based on 18S rRNA and COI gene sequences, supporting monophyly of the two morphospecies. However, ITS sequences did not resolve these taxa (Fig 3). The number of species within L. melicerta ranged from 9–30 based on COI and 6–45 based on ITS region sequences. For L. ceratophylli, 3–10 species were identified based on COI gene, and 3–20 based on ITS region sequences (Figs 2 and 3; Table 2). The most conservative results were nine putative species for L. melicerta (BSD based on COI gene sequences) and three species for L. ceratophylli (*BEAST based on COI gene sequences). However, *BEAST classified a distinct clade that was represented by a population from Florida as part of species D. That clade was considered a separate species by BSD and ABGD (Fig 2). As *BEAST may have underestimated diversity within L. ceratophylli, we delimited cryptic species based on BSD analysis of the COI gene sequences, which was the second most conservative method for this species (Table 2).
Average branch lengths are proportional to the number of substitutions per site under a TPM2uF+I+G substitution model. At each node posterior probabilities > 0.80 are shown. Putative cryptic species detected using Bayesian Species Delimitation (BSD), *BEAST, Automatic Barcoding Gap Discovery (ABGD), and Single Threshold Generalized Mixed Yule Coalescent models (GMYC) are shown. Abbreviations as in S1 and S2 Tables; independent clonal isolates are indicated by a number (e.g. 01).
Average branch lengths are proportional to the number of substitutions per site under a TPM1uF+I substitution model. Posterior probabilities > 0.80 are shown at nodes. Putative cryptic species found using Bayesian Species Delimitation (BSD), *BEAST, Automatic Barcoding Gap Discovery (ABGD), and Single Threshold Generalized Mixed Yule Coalescent models (GMYC) are shown. Abbreviations as in S1 and S2 Tables; independent clonal isolates are indicated by a number (e.g. 01).
The mean genetic distance in COI gene sequences was 0–15.4% within cryptic species of L. melicerta, and 0.5–0.6% within cryptic species of L. ceratophylli (BSD based on COI gene sequences). Among BSD cryptic species, the COI mean genetic distance was 8.1–21.9% for L. melicerta, and 15.1–21.2% for L. ceratophylli. For ITS region sequences, within cryptic species mean genetic distance ranged 0.08–2.3% for L. melicerta and 0.04–1.5% for L. ceratophylli. Among putative cryptic species, the ITS mean genetic distance was 0–2% for L. melicerta, and 0–4.3% for L. ceratophylli. In the haplotype networks based on COI gene sequences for both species, genotype clusters corresponded to the BSD cryptic species (Fig 4).
Size of circles is proportional to the number of sequences sharing the same haplotype. Branch lengths are proportional to the number of nucleotide substitutions. Black symbols on the network correspond to cryptic species detected by Bayesian Species Delimitation. Color codes are based on the collection site; Texas: red, New Mexico: light green, Georgia: purple, Oregon: light blue, Utah: brown, Oklahoma: gray, California: pink, Florida: dark blue, Wisconsin: dark green, New Hampshire: yellow, Carolina: orange, Pennsylvania: salmon, Minnesota: gold, Australia: dark pink, outgroups: white.
There was discordance between phylogenetic trees based on COI gene and ITS region sequences. For example, COI lineages E, F, G and H are clustered as one lineage based on the ITS region (lineage 8). In another example, one lineage (lineage 9) based on the ITS region is composed of populations from multiple COI cryptic species (E, I and M) (Figs 2 and 3).
Isolation by distance
For Limnias melicerta populations there was a significant, but weak, correlation between genetic distance and log transformed geographic distance for both markers (Mantel test: COI: r = 0.4 with 95% confidence interval of 0.27–0.46, p < 0.001 and ITS: r = 0.14 with 95% confidence interval of 0.06–0.2, p = 0.03). For L. ceratophylli, genetic variation in the COI gene was significantly correlated to log transformed geographic distance (Mantel test: r = 0.3; 95% confidence interval of 0.27–0.4, p = 0.001). However, this correlation was not significant based on ITS sequences (Mantel test: r = -0.02; 95% confidence interval of -0.12 to 0.08, p = 0.8).
Trophi morphology
We obtained 92 SEM images of trophi for L. melicerta representing six putative species, and 60 images for L. ceratophylli representing two cryptic species based on COI sequences. Trophi images are available at UTEP Bioinformatics Data Repository. Representative trophi images for each cryptic species are shown in S2 Fig. No significant variation was detected between L. melicerta and L. ceratophylli in trophi shape (Discriminant Analysis: frontal: Wilks’ Lambda = 1, Chi-square < 0.001, df = 16, p = 1, and caudal: Wilks’ Lambda = 1, Chi-square = 0.001, df = 42, p = 1) or trophi size (frontal: Mann-Whitney U = 911, p = 0.76, and caudal: Mann-Whitney U = 298, p = 0.77). Trophi shape did not show significant variation among putative species of L. melicerta (Discriminant Analysis: frontal: Wilks’ Lambda = 1, Chi-square = 0.001, df = 80, p = 1, and caudal: Wilks’ Lambda = 1, Chi-square = 0.0, df = 70, p = 1) or L. ceratophylli (Discriminant Analysis: frontal: Wilks’ Lambda = 1, Chi-square = 0.001, df = 12, p = 1, and caudal: Wilks’ Lambda = 1, Chi-square = 0.001, df = 28, p = 1). There was significant variation in trophi size among putative species of L. melicerta (frontal: Kruskal-Wallis Chi-square = 12.2, p = 0.002, and caudal: Kruskal-Wallis Chi-square = 10.3, p = 0.02). Cryptic species D had the smallest centroid size (frontal and caudal: 1.33) and cryptic species E had the largest centroid size (frontal: 4, and caudal: 5). However, trophi size showed no significant differentiation among putative species of L. ceratophylli (frontal: Kruskal-Wallis Chi-square = 2.5, p = 0.11, and caudal: Kruskal-Wallis Chi-square = 5.9, p = 0.051).
Discussion
With the advent of molecular tools, detecting cryptic species in rotifers has become a common occurrence, which has improved our knowledge of their diversity. All the previous studies about genetic variation and cryptic species in rotifers are on non-sessile taxa. Here, using two molecular markers we found cryptic diversity within two morphospecies of sessile rotifers, Limnias melicerta and L. ceratophylli. Using the BSD delimitation method, nine putative cryptic species for L. melicerta and four putative cryptic species for L. ceratophylli were identified based on COI gene sequences.
Although benefits of using multiple markers to investigate cryptic species is recognized [100–104], most studies of rotifers are based solely on the mitochondrial COI gene (e.g., [24–27,105–107]). In their study of the Brachionus plicatilis complex based on all available COI and ITS sequences, Mills et al. [29] recommended the nuclear marker ITS1 over COI as it gave a more conservative estimate of species diversity. Similarly, the ITS region was recommended for species delimitation within the Euchlanis dilatata complex by Kordbacheh et al. [28]. In another study, Papakostas et al. [30] reported discordance between COI and ITS markers for species delimitation within the Brachionus calyciflorus complex. They argued that species delimitation based on the ITS region better explains morphological variation within the complex. In studies of other taxa, mitochondrial and nuclear markers are concatenated for species delimitation (e.g., bothriurid scorpions Brachistosternus spp. complexes [108]; copepod Cyclops spp. complexes: [109]), or nuclear markers are used to complement species delimitations which were based on mitochondrial markers (e.g., the sea spider Pallenopsis patagonica complex (Hoek, 1881) [110]; Madagascar’s Mouse Microcebus spp. complexes [111]). We did not use a concatenated dataset for species delimitation because of differences in coalescent times for these markers [112], and discordance between COI and ITS phylogenetic trees. The observed discordance indicates a lack of coherence within cryptic species for mitochondrial and nuclear markers. This could be due to introgression as was shown for B. calyciflorus [30] or incomplete lineage sorting as in E. dilatata [28], Keratella cochlearis Gosse, 1851, Polyarthra dolichoptera Idelson, 1925, and Synchaeta pectinata Ehrenberg, 1832 [113]. In our study, species delimitation based on the ITS region was more conservative as compared to the COI gene. However, the ITS region showed overall low levels of variation (L. melicerta: ≤3.1%, L. ceratophylli: ≤4.4%) and failed to separate L. melicerta and L. ceratophylli in the phylogenetic analysis. These traditionally recognized species were monophyletic based on COI and 18S rRNA gene sequences. Also, mitochondrial genes can be more informative in the phylogenetic analyses of recently diverged lineages because of their faster rate of evolution [114]. These markers have been successfully used to delimit species in, for example, the Puerto Rican termite Heterotermes [115], the copepod Paracalanus parvus complex [116], and within several rotifer species (e.g., K. cochlearis [117]; S. pectinata [24]; P. dolichoptera [26]; E. senta [37]; Lecane bulla (Gosse, 1851) [118]). Therefore despite the possibility of over-splitting, we used COI gene sequences for species delimitation within L. melicerta and L. ceratophylli.
Populations included in this study were obtained from a wide geographic range. For example, L. melicerta populations came from Oregon (USA) and the state of Victoria (Australia) a distance of 12,890 km; and L. ceratophylli populations came from sites spanning from Oregon and Georgia, a distance of 2,726 km. Cryptic species showed various distribution ranges, from a single habitat to several distant habitats. For example, L. melicerta H was collected only from a permanent lake in Florida, while K was collected from water bodies in New Mexico, Oklahoma, Texas, and Florida. Of the four L. ceratophylli cryptic species, two were collected from variety of habitats across our sampling range. L. ceratophylli B was found in sites in Texas, Wisconsin, Oregon, Oklahoma, Pennsylvania, Minnesota, and species D from Georgia, California, and Pennsylvania. The other two L. ceratophylli cryptic species are singletons, one from a permanent lake in New Mexico, and the other from a pond in Florida. The observed variation in geographic distributions of these putative species may be an artifact of under sampling. The geographic distributions of these cryptic species may be expanded by including samples from additional biogeographical regions or they may represent lineages unique to a given locality. There is also the possibility that some cryptic species are, in fact, cosmopolitan. However, similar patterns in distribution have been reported for other rotifer species complexes such as E. dilatata (two singletons, five widely distributed species; [28]) and B. plicatilis (SM7 restricted to North America, SM8 to Australia, and B. ibericus Ciros-Pérez, Gómez & Serra, 2001 to Europe; [29]). We observed cryptic species represented by few populations that showed wide geographic ranges; as noted above cryptic species G from L. melicerta was represented by two clonal isolates; one found in Oregon and the other in Georgia, and cryptic species I comprised four populations from Wisconsin, Oregon and Utah. Thus, cryptic species in our study probably vary in their ability for dispersal and colonizing distant habitats. Intraspecific variation in dispersal ability that we infer here has been discussed for other passively dispersing taxa (see [119]).
In our study, genetic variation among populations based on COI gene and ITS region sequences was significantly correlated with geographic distance, except for ITS sequences corresponding to L. ceratophylli populations. However, because the correlation was weak (or lacking), geographic isolation cannot be considered a strong driver of genetic variation within these cryptic species. Similar to studies on other invertebrates (e.g., 18 invertebrate species [120]; Daphnia lumholtzi [121]; E. dilatata [28]; S. pectinata [24]; B. calyciflorus [122]), the populations studied here showed high genetic differentiation across small geographic scales.
We compared the genetic divergence estimates for four sessile rotifer species with non-sessile rotifers. There is evidence of habitat preference in sessile and non-sessile littoral species (e.g., Collotheca campanulata [123] and E. dilatata [44], respectively). As mentioned above, this differentiation can contribute to genetic divergence among populations. Therefore, we expected to see similar levels of genetic differentiation for sessile and littoral morphospecies and lower genetic divergence for planktonic rotifers. Examples of genetic divergence in ITS region and COI gene in rotifer species with different lifestyles are summarized in Table 3. As shown in Table 3, genetic variation in ITS region sequences for littoral rotifer species (E. dilatata and L. bulla) was higher than that of planktonic and sessile species complexes. Besides, genetic variation in ITS region in sessile species was similar to that of planktonic groups. It should be noted that there are few instances where the ITS region is used to study rotifer cryptic species and reporting levels of differentiation among cryptic lineages (Table 3). Therefore, there may not be sufficient information to make conclusions about the relationship between rotifer life style and genetic differentiation in the ITS region. On the other hand, higher diversification rates in the COI gene was reported in bdelloid rotifers compared to monogononts [40]. It is hypothesized that this difference is related to the difference in reproductive mode between bdelloids and monogononts (obligatory v. facultative parthenogenic, respectively) [40]. In this study, genetic variation in the COI gene for L. melicerta and L. ceratophylli was similar to some other non-rotifer sessile taxa (Table 3). However, genetic variation in the COI gene among rotifer cryptic species was within the same range for planktonic, littoral, and sessile groups (Table 3). A similar pattern has been reported for marine nematodes where genetic structure was not related to their habitat preference (algae versus sediments) [124]. It should be noted that within the superorder Gnesiotrocha, there is no difference in levels of genetic variation between different life styles (at least for the taxa examined thus far; sessile: Limnias spp. and Collotheca spp. and littoral: Testudinella clypeata Müller, 1786, Table 3). All of these rotifers are cyclical parthenogens, which may have contributed to similarity in the range of genetic variation within them. Comparing diversification rate among rotifers with different life history features may not be possible by simply considering the genetic variation in COI gene. To investigate the patterns of diversification for different groups of rotifers, more sophisticated statistical analyses are essential. For example, Fontaneto et al. [40] used a statistic measure (γ) to compare the relative position of nodes in the phylogeny to the positions expected under a constant diversification rate scenario. Using this approach, positive values of γ show that diversification rate is higher than expected. γ was used to compare diversification rate among taxa. In another study, Fontanillas et al. [125] calculated branch length in the phylogenetic tree and used it to compare diversification rate between sister taxa.
Sometimes morphological variation among cryptic species is found after a more detailed analysis (e.g., [37,130]). Thus, we investigated trophi morphology to identify potential variation among cryptic species of L. melicerta and L. ceratophylli. Limnias possess malleoramate trophi. This type of trophi, has large teeth on the rami and thin teeth on the unci [50]. For more detailed descriptions of Limnias trophi, see Gosse [59], Meksuwan et al. [51] and Wallace et al. [54]. While trophi size differed among putative cryptic species of L. melicerta, it did not show significant variation among putative cryptic species of L. ceratophylli or between the two. The high variability in trophi size within each morphospecies may have led to failure in detecting significant differences between them. No significant variation in trophi shape was found between L. melicerta and L. ceratophylli, or among putative cryptic species. Therefore, trophi shape cannot be used as a diagnostic character in distinguishing them. This morphological conservation and stasis in trophi morphology, despite high genetic variation among putative cryptic species (COI gene: L. melicerta ≤ 22.7%, L. ceratophylli ≤ 21.7%), may potentially stem from incongruence between rates of speciation and morphological evolution [131]. Morphological stasis in a variety of traits has been observed in numerous organisms (e.g., the copepod Eurytemora affinis (Poppe, 1880) [131]; the butterfly fish Pantodon buchholzi Peters, 1876 [132]; two amphipods Leucothoe ashleyae Thomas and Klebba, 2006 and Leucothoe kensleyi Thomas and Klebba, 2005 [133]). Differences in trophi morphology has been shown to be connected to variation in feeding habits (see Asplanchnidae [134]). Therefore, morphological stasis in trophi could be a result of ecological niche conservatism through similarity in the diet of L. melicerta and L. ceratophylli, both of which feed on small particles including yeast [135] and planktonic algae [64]. Morphological stasis should be investigated within morphospecies of other rotifer taxa such as the genus Floscularia. This is because, unlike L. melicerta and L. ceratophylli, Floscularia species show interspecies variation in trophi morphology [136]. Thus, they are likely to show variation in trophi features at the level of cryptic lineages.
Similar to other taxonomic groups, there are a variety of studies that did not find significant morphological variation among rotifer cryptic species. Based on geometric morphometric analyses of lorica and trophi features, Fontaneto et al. [130] suggested there are no robust morphological differences between B. plicatilis and B. manjavacas. In addition, Leasi et al. [25] did not find any significant variation in morphological features of the lorica or body size among seven cryptic species of the T. clypeata complex. Similarly, morphological characteristics such as variation in lorica size and the presence of a posterior spine were not able to distinguish among eight putative cryptic species of K. cochlearis [117]. In summary, morphological features have not always been effective in distinguishing among cryptic species of rotifers.
Several studies have complemented molecular methods with other types of data to delimit species boundaries (e.g., [30,106,122,130,137–139]). In this study, we attempted to use a morphological characteristic and geographic isolation of genotypes to find a reliable predictor of genetic variation among putative species within Limnias melicerta and L. ceratophylli. However, trophi shape did not vary among cryptic species, and there was no strong correlation between genetic and geographic distance in these species. Therefore, we did not find sufficient morphological variation or geographic isolation needed to explain the observed genetic differentiation among them. Past studies have focused on ecological differences between L. melicerta and L. ceratophylli. For instance, Sarma et al. [64] showed that generation time changes in response to food concentration for L. ceratophylli, while there is no effect on L. melicerta. While ecological differentiation has been recorded among many rotifer cryptic species (see [140]), to our knowledge there is no information on ecological or behavioral variation among genetic entities of any Limnias species. Therefore, we recommend further examination of these features in Limnias spp. to elucidate potential mechanisms involved in their speciation. To examine ecological and behavioral differentiation among cryptic species, future studies should include more comprehensive morphological analyses, life table and mating experiments, investigating patterns of metamorphosis and substrate selection by larvae. Studies such as these will aid in examining boundaries of sessile rotifer cryptic species and understanding speciation in rotifers. Moreover, genetic differentiation in other sessile rotifers should be measured to obtain more data for comparing diversification rate among taxa with different life styles (e.g., planktonic versus sessile, colonial versus solitary).
Supporting information
S1 Table. Site and date of collection of Limnias melicerta populations and outgroup taxa.
GenBank accession numbers for their corresponding partial COI gene, ITS region, and partial 18S rRNA sequences are provided. Number of sequenced clonal lineages and haplotype group(s) for each population are also noted. Missing sequences are specified by “-” for haplotype group and GenBank accession numbers.
https://doi.org/10.1371/journal.pone.0205203.s001
(DOCX)
S2 Table. Site and date of collection of Limnias ceratophylli populations.
GenBank accession numbers for their corresponding partial COI gene, ITS region, and partial 18S rRNA sequences are provided. Number of sequenced clonal lineages and haplotype group(s) for each population are also noted. Missing sequences are specified by “-” for haplotype group and GenBank accession numbers. Outgroups are the same as in S1 Table.
https://doi.org/10.1371/journal.pone.0205203.s002
(DOCX)
S3 Table. Substitution saturation test of molecular markers.
Substitution saturation test for partial COI gene, ITS region, and partial 18S rRNA sequences of Limnias melicerta and L. ceratophylli populations implemented in DAMBE v 6. Iss: index of substitution saturation, and Iss.c: critical index of substitution saturation. If Iss is significantly smaller than Iss.c, there is little saturation in the sequences.
https://doi.org/10.1371/journal.pone.0205203.s003
(DOCX)
S1 Fig. Bayesian inference consensus phylogenetic tree based on partial 18S rRNA sequences of 17 populations of Limnias melicerta and 18 populations of L. ceratophylli.
Average branch lengths are proportional to the number of substitutions per site under a JC substitution model. At each node, posterior probabilities > 0.80 are shown. Abbreviations as in S1 and S2 Tables.
https://doi.org/10.1371/journal.pone.0205203.s004
(PDF)
S2 Fig. Representative trophi from cryptic species of Limnias melicerta and L. ceratophylli.
Trophi of cryptic species E, G, I, K, L and M from L. melicerta and trophi of cryptic species B and D from L. ceratophylli are shown.
https://doi.org/10.1371/journal.pone.0205203.s005
(DOCX)
Acknowledgments
We offer our special thanks to Elizabeth Preza and UTEP’s BBRC Genomic Analysis Core Facility for their assistance in obtaining and analyzing molecular data. Rick Hochberg, Carl Lieb, Kevin Floyd, Patrick Brown, Jennifer Martinez, Jennifer Apodaca, Karla Garcia, Priscilla Duran, and Jose Rivas, Jr. provided water samples. Rochelle Petrie and Russ Shiel collected sediment samples in Australia. The department of Metallurgical, Materials and Biomedical Engineering at the University of Texas at El Paso provided access to the scanning electron microscope. Our manuscript has benefited from insightful comments provided by Hendrik Segers, Michael Moody, Arshad Khan, and two anonymous reviewers. Rotifers were collected under permits TPWD 2014–01 (E.J. Walsh), TPWD 2015–03 (E. Walsh), OPRD-015-14 (E.J. Walsh), CPDCNBSP-2016-32 (P.D. Brown), CAVE-2012-SCI-0001 (E.J. Walsh), City of Los Angeles Department of Recreation and Parks (E.J. Walsh), Utah Department of Natural Resources (E.J. Walsh), Energy, Minerals and Natural Resources Department State Parks Division (E.J. Walsh).
References
- 1. Finlay BJ. Global dispersal of free-living microbial eukaryote species. Science. 2002;296: 1061–1063. pmid:12004115
- 2. Fenchel T. Cosmopolitan microbes and their cryptic species. Aquat Microb Ecol. 2005;41: 49–54.
- 3. Teittinen A, Soininen J. Testing the theory of island biogeography for microorganisms—patterns for spring diatoms. Aquat Microb Ecol. 2015;75: 239–250. http://www.int-res.com/abstracts/ame/v75/n3/p239-250.
- 4. Mazaris AD, Moustaka-Gouni M, Michaloudi E, Bobori DC. Biogeographical patterns of freshwater micro- and macroorganisms: a comparison between phytoplankton, zooplankton and fish in the eastern Mediterranean. J Biogeogr. 2010;37: 1341–1351.
- 5. Fierer N, Jackson RB. The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci. 2006;103: 626–631. pmid:16407148
- 6. Schauer R, Bienhold C, Ramette A, Harder J. Bacterial diversity and biogeography in deep-sea surface sediments of the South Atlantic Ocean. ISME J. 2010;4: 159–70. pmid:19829317
- 7.
Marteinsson VÞ, Groben R, Reynisson E, Vannier P. Biogeography of marine microorganisms. In: Stal LJ, Cretoiu MS, editors. The marine microbiome: An untapped source of biodiversity and biotechnological potential. Springer International Publishing, 2016, pp. 187–207. https://doi.org/10.1007/978-3-319-33000-6_6
- 8. Salerno JL, Bowen BW, Rappé MS. Biogeography of planktonic and coral-associated microorganisms across the Hawaiian Archipelago. FEMS Microbiol Ecol. 2016;92;http://dx.doi.org/10.1093/femsec/fiw109.
- 9. Chao A, Li PC, Agatha S, Foissner W. A statistical approach to estimate soil ciliate diversity and distribution based on data from five continents. Oikos. 2006;114: 479–493.
- 10. Foissner W, Chao A, Katz LA. Diversity and geographic distribution of ciliates (Protista: Ciliophora). Biodivers Conserv. 2008;17: 345–363.
- 11. Gumiere T, Durrer A, Bohannan BJM, Andreote FD. Biogeographical patterns in fungal communities from soils cultivated with sugarcane. J Biogeogr. 2016;43: 2016–2026.
- 12. Dumont HJ. Biogeography of rotifers. Hydrobiologia. 1983; 104: 19–30.
- 13. Segers H. The biogeography of littoral Lecane Rotifera. Hydrobiologia. 1996;323: 169–197.
- 14.
Artois T, Fontaneto D, Hummon W, McInnes S, Todaro A, Sorenson M, et al. Ubiquity of micoscopic animals? Evidence from the morphological approach in species identification. In: Fontaneto D, editor. Biogeography of microscopic organisms. Is everything small everywhere? Cambridge University Press, 2011, pp. 244–283.
- 15.
Guil N. Molecular approach to micrometazoans. Are they here, there and everywhere? In: Fontaneto D, editor. Biogeography of microscopic organisms. Is everything small everywhere? Cambridge University Press, 2011, pp. 284–306.
- 16. Hahn MW, Jezberova J, Koll U, Saueressig-Beck T, Schmidt J. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences. ISME J. 2016;10: 1642–1655. http://dx.doi.org/10.1038/ismej.2015.237 pmid:26943621
- 17. Kon T, Yoshino T, Mukai T, Nishida M. DNA sequences identify numerous cryptic species of the vertebrate: A lesson from the gobioid fish Schindleria. Mol Phylogenet Evol. 2007;44: 53–62. pmid:17275344
- 18. Lahr DJG, Laughinghouse HD, Oliverio AM, Gao F, Katz LA. How discordant morphological and molecular evolution among microorganisms can revise our notions of biodiversity on Earth. Int J BioEssays. 2014;36: 950–959. pmid:25156897
- 19. Šlapeta J, Moreira D, López-García P. The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes. Proc Biol Sci. 2005;272: 2073–2081. http://rspb.royalsocietypublishing.org/content/272/1576/2073.abstract. pmid:16191619
- 20.
Wallace RL, Nogrady T, Snell TW, Ricci C. Rotifera Volume 1: Biology, ecology and systematics. 2nd ed. Nogrady T, editor. Kenobi Productions, 2006, 293 pp.
- 21. Walsh EJ, May L, Wallace RL. A metadata approach to documenting sex in phylum Rotifera: diapausing embryos, males, and hatchlings from sediments. Hydrobiologia. 2016;796: 265–276.
- 22. Rousselet CF. On the geographic distribution of the Rotifera. Microscopy. 1909;10: 465–470.
- 23. Fontaneto D, Iakovenko N, Eyres I, Kaya M, Wyman M, Barraclough TG. Cryptic diversity in the genus Adineta Hudson & Gosse, 1886 (Rotifera: Bdelloidea: Adinetidae): a DNA taxonomy approach. Hydrobiologia. 2011;662: 27–33.
- 24. Kimpel D, Gockel J, Gerlach G, Bininda-Emonds ORP. Population structuring in the monogonont rotifer Synchaeta pectinata: high genetic divergence on a small geographical scale. Freshw Biol. 2015;60: 1364–1378.
- 25. Leasi F, Tang CQ, De Smet WH, Fontaneto D. Cryptic diversity with wide salinity tolerance in the putative euryhaline Testudinella clypeata (Rotifera, Monogononta). Zool J Linn Soc. 2013;168: 17–28.
- 26. Obertegger U, Flaim G, Fontaneto D. Cryptic diversity within the rotifer Polyarthra dolichoptera along an altitudinal gradient. Freshw Biol. 2014;59: 2413–2427.
- 27. Wen X, Xi Y, Zhang G, Xue Y, Xiang X. Coexistence of cryptic Brachionus calyciflorus (Rotifera) species: roles of environmental variables. J Plankton Res. 2016;38: 478–489.
- 28. Kordbacheh A, Garbalena G, Walsh EJ. Population structure and cryptic species in the cosmopolitan rotifer Euchlanis dilatata. Zool J Linn Soc. 2017;181: 757–777.
- 29. Mills S, Alcántara-Rodríguez JA, Ciros-Pérez J, Gómez A, Hagiwara A, Galindo KH, et al. Fifteen species in one: deciphering the Brachionus plicatilis species complex (Rotifera, Monogononta) through DNA taxonomy. Hydrobiologia. 2016;796: 39–58.
- 30. Papakostas S, Michaloudi E, Proios K, Brehm M, Verhage L, Rota J, et al. Integrative taxonomy recognizes evolutionary units despite widespread mitonuclear discordance: Evidence from a rotifer cryptic species complex. Syst Biol. 2016;65: 508–524. http://dx.doi.org/10.1093/sysbio/syw016 pmid:26880148
- 31. Fu Y, Hirayama K, Natsukari Y. Morphological differences between two types of the rotifer Brachionus plicatilis O.F. Müller. J Exp Mar Bio Ecol. 1991;151: 29–41. http://dx.doi.org/10.1016/0022-0981(91)90013-M.
- 32. Anitha PS, George RM. The taxonomy of Brachionus plicatilis species complex (Rotifera: Monogononta) from the Southern Kerala (India) with a note on their reproductive preferences. J Mar Biol Assoc India. 2006;48: 6–13.
- 33. Campillo S, García-Roger EM, Martínez-Torres D, Serra M. Morphological stasis of two species belonging to the L-morphotype in the Brachionus plicatilis species complex. Hydrobiologia. 2005;546: 181–187.
- 34. Ciros-Pérez J, Gómez A, Serra M. On the taxonomy of three sympatric sibling species of the Brachionus plicatilis (Rotifera) complex from Spain, with the description of B. ibericus n. sp. J Plankton Res. 2001;23: 1311–1328.
- 35. Hwang DS, Dahms HU, Park H, Lee JS. A new intertidal Brachionus and intrageneric phylogenetic relationships among Brachionus as revealed by allometry and CO1-ITS1 gene analysis. Zool Stud. 2013;52: 13.
- 36. Michaloudi E, Mills S, Papakostas S, Stelzer CP, Triantafyllidis A, Kappas I, et al. Morphological and taxonomic demarcation of Brachionus asplanchnoidis Charin within the Brachionus plicatilis cryptic species complex (Rotifera, Monogononta). Hydrobiologia. 2016;796: 19–37.
- 37. Schröder T, Walsh EJ. Cryptic speciation in the cosmopolitan Epiphanes senta complex (Monogononta, Rotifera) with the description of new species. Hydrobiologia. 2007;593: 129–140.
- 38. Fontaneto D, Herniou EA, Barraclough TG, Ricci C, Melone G. On the reality and recognisability of asexual organisms: morphological analysis of the masticatory apparatus of bdelloid rotifers. Zool Scr. 2007;36: 361–370.
- 39. Eastman JM, Storfer A. Correlations of life-history and distributional-range variation with salamander diversification rates: evidence for species selection. Syst Biol. 2011;60: 503–518. http://dx.doi.org/10.1093/sysbio/syr020 pmid:21460386
- 40. Fontaneto D, Tang CQ, Obertegger U, Leasi F, Barraclough TG. Different diversification rates between sexual and asexual organisms. Evol Biol. 2012;39: 262–270.
- 41. Fluker B, Kuhajda B, Harris P. The influence of life‐history strategy on genetic differentiation and lineage divergence in darters (Percidae: Etheostomatinae). Evolution. 2014;68: 3199–3216. pmid:25130551
- 42. Takeda M, Kusumi J, Mizoiri S, Aibara M, Mzighani SI, Sato T, et al. Genetic structure of pelagic and littoral cichlid fishes from Lake Victoria. PLoS One. 2013;8; https://doi.org/10.1371/journal.pone.0074088.
- 43. Segers H. Annotated checklist of the rotifers (Phylum Rotifera), with notes on nomenclatures, taxonomy and distribution. Zootaxa. 2007;1564: 1–104.
- 44. Wallace RL, Edmondson WT. Mechanism and adaptive significance of substrate selection by a sessile rotifer. Ecology. 1986;67: 314–323.
- 45. Walsh EJ. Oviposition bahavior of the litoral rotifer Euchlanis dilatata. Hydrobiologia. 1989;186/187: 157–161.
- 46. Derycke S, Backeljau T, Vlaeminck C, Vierstraete A, Vanfleteren J, Vincx M, et al. Seasonal dynamics of population genetic structure in cryptic taxa of the Pellioditis marina complex (Nematoda: Rhabditida). Genetica. 2006;128: 307–321. pmid:17028960
- 47. Burton RS, Feldman MW. Population genetics of Tigriopus californicus. II. Differentiation among neighboring populations. Evolution. 1981;35: 1192–1205. pmid:28563405
- 48. Russo CAM, Solé-Cava AM, Thorpe JP. Population structure and genetic variation in two tropical sea anemones (Cnidaria, Actinidae) with different reproductive strategies. J Mar Biol. 1994;119: 267–276.
- 49. Lee PLM, Dawson MN, Neill SP, Robins PE, Houghton JDR, Doyle TK, et al. Identification of genetically and oceanographically distinct blooms of jellyfish. J R Soc Interface. 2013;10: http://rsif.royalsocietypublishing.org/content/10/80/20120920.
- 50. Stopar K, Ramšak A, Trontelj P, Malej A. Lack of genetic structure in the jellyfish Pelagia noctiluca (Cnidaria: Scyphozoa: Semaeostomeae) across European seas. Mol Phylogenet Evol. 2010;57: 417–428. https://doi.org/10.1016/j.ympev.2010.07.004 pmid:20637295
- 51.
Wallace RL, Snell TW. Chapter 8: Rotifera. Thorp JH, Covich A, editors. Academic Press, 2010, pp. 173–235. https://doi.org/10.1016/B978-0-12-374855-3.
- 52. Meksuwan P, Pholpunthin P, Segers H. Molecular phylogeny confirms Conochilidae as ingroup of Flosculariidae (Rotifera, Gnesiotrocha). Zool Scr. 2015; 44: 562–573.
- 53. Sørensen MV, Giribet G. A modern approach to rotiferan phylogeny: combining morphological and molecular data. Mol Phylogenet Evol. 2006; 40: 585–608. pmid:16690327
- 54.
Jersabek CD, De Smet WH, Fischer C, Fontaneto D, Michaloudi E, Wallace RL, Segers H. Candidate Rotifera part of the List of Available Names in Zoology. 2011. http://rotifera.hausdernatur.at/Home/RotiferLAN. Accessed on 09/18/2018.
- 55. Wallace RL, Kordbacheh A, Walsh EJ. Key to the currently recognized species of Limnias Schrank, 1803 (Rotifera, Monogononta, Gnesiotrocha, Flosculariidae). Zootaxa. 2018;4442: 307–318.
- 56. Rousselet CF. Note on a new rotifer, Limnias Cornuella. Microscopy. 1889;3: 337–338.
- 57. Cubitt C. A rare Melicertian; with remarks on the homological position of this form, and also on a previously-recorded new species Floscularia coronetta. Mon Microsc J. 1871;6: 165–170.
- 58. Tatem J. On a new Melicertian and some varieties of Melicerta ringens. JMicroscopy. 1868;1: 124–125.
- 59. Wright H. The ringed tube of Limnias melicerta Weisse. Microscope. 1954;10: 13–19.
- 60. Gosse P. On the structure, functions, and homologies of the manducatory organs in the class Rotifera. Philos Trans R Soc Lond B Biol Sci. 1856;146: 419–452.
- 61. Yang H, Hochberg R. Ultrastructure of the extracorporeal tube and cement glands in the sessile rotifer Limnias melicerta (Rotifera: Gnesiotrocha). Zoomorphology. 2018;137: 1–12.
- 62. Kutikova LA. Larval metamorphosis in sessile rotifers. Hydrobiologia. 1995;313: 133–138.
- 63. Bankit S. Sessile rotifer species Limnias melicerta as bioindicator of non-stressed water body. Environ Ecol. 1995;13: 56–58.
- 64. Arora J, Mehra NK. Species diversity of planktonic and epiphytic rotifers in the backwaters of the Delhi segment of the Yamuna river, with remarks on new records from India. Zool Stud. 2003;42: 239–247.
- 65. Sarma SSS, Jiménez-Santos MA, Nandini S, Wallace RL. Demography of the sessile rotifers, Limnias ceratophylli and Limnias melicerta (Rotifera: Gnesiotrocha), in relation to food (Chlorella vulgaris Beijerinck, 1890) density. Hydrobiologia. 2017;796: 181–189.
- 66. Upreti N, Sharma S, Sharma S, Sharma KP. Acute and chronic toxicity of fluoride and aluminum on plant and animal models. Res Rev A J Toxicol. 2012;1: 1–17.
- 67. Stemberger RS. A general approach to the culture of planktonic rotifers. Can J Fish Aquat Sci. 1981;38: 721–724.
- 68. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3: 294–299. pmid:7881515
- 69.
White T, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Shinsky J, White T, editors. PCR Protocols: A guide to methods and applications. Academic Press, 1990, pp. 315–322.
- 70. Giribet G, Carranza S, Baguna J, Riutort M, Ribera C. First molecular evidence for the existence of a Tardigrada + Arthropoda clade. Mol Biol Evol. 1996;13: 76–84. pmid:8583909
- 71. Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC. The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Syst Biol. 1997;46: 1–68. pmid:11975347
- 72.
Geospiza.com. FinchTV. Seattle, WA; 2014: http://www.geospiza.com/Products/finchtv.shtml.
- 73. Flot JF. seqphase: a web tool for interconverting phase input/output files and fasta sequence alignments. Mol Ecol Resour. 2010;10: 162–166. pmid:21565002
- 74. Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9: 868–877. pmid:10508846
- 75. Katoh K, Standley DM. MAFFT. Multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30: 772–780. pmid:23329690
- 76.
Maddison WP, Maddidon DR. Mesquite: a modular system for evolutionary analysis. 2017: Version 3.2. http://mesquiteproject.org.
- 77. Xia X. DAMBE6: New tools for microbial genomics, phylogenetics and molecular evolution. J Hered. 2017;108: 431–437. pmid:28379490
- 78. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25: 1451–1452. pmid:19346325
- 79. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2015;33: 1870–4. pmid:24132122
- 80. Bandelt HJ, Forster P, Rohl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999;16: 37–48. pmid:10331250
- 81. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9: 772. http://dx.doi.org/10.1038/nmeth.2109.
- 82. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52: 696–704. pmid:14530136
- 83.
Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES science gateway for inference of large phylogenetic trees. 2010. Gateway Computing Environments Workshop, 2010, pp. 1–8.
- 84. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012;29: 1969–1973. pmid:22367748
- 85. Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S, et al. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol. 2006;55: 595–609. pmid:16967577
- 86. Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinformatics. 2013;29: 2869–76. pmid:23990417
- 87. Puillandre N, Lambert A, Brouillet S, Achaz G. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol. 2012;21: 1864–1877. pmid:21883587
- 88. Rannala B, Yang Z. Bayes estimation of species divergence times and ancestral population sizes using DNA sequences from multiple loci. Genetics. 2003;164: 1645–56. pmid:12930768
- 89. Yang Z, Rannala B. Bayesian species delimitation using multilocus sequence data. Proc Natl Acad Sci. 2010;107: 9264–9269. pmid:20439743
- 90. Yang Z, Rannala B. Unguided species delimitation using DNA sequence data from multiple loci. Mol Biol Evol. 2014;31: 3125–3135. pmid:25274273
- 91.
Rambaut A, Suchard MA, Xie D, Drummond AJ. Tracer v1.6. http://beast.bio.ed.ac.uk/Tracer.
- 92. Fujisawa T, Barraclough TG. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: a revised method and evaluation on simulated data sets. Syst Biol. 2013; 62: 707–724. pmid:23681854
- 93. Reid NM, Carstens BC. Phylogenetic estimation error can decrease the accuracy of species delimitation: a Bayesian implementation of the general mixed yule coalescent model. BMC Evol Biol. 2012;12: 196. pmid:23031350
- 94.
Ersts PJ. Geographic distance matrix generator version 1.2.3. Nat Hist. 2006; http://biodiversityinformatics.amnh.org/open_source/gdmg/documentation.php.
- 95. Goslee SC, Urban DL. The ecodist package for dissimilarity-based analysis of ecological data. J Stat Software. 2007;22: 1–19. https://www.jstatsoft.org/v022/i07.
- 96. Segers H. Rotifera of some lakes in the floodplain of the River Niger (Imo State, Nigeria). New species and other taxonomic considerations. Hydrobiologia. 1993;250: 39–61.
- 97. Adams DC, Rohlf FJ, Slice DE. Geometric morphometrics: Ten years of progress following the revolution. Ital J Zool. 2004;71: 5–16.
- 98. Rohlf FJ. The tps series of software. Hystrix. 2015;26: 1–4.
- 99. Cavalcanti MJ, Monteiro LR, Lopes PRD. Landmark-based morphometric analysis in selected species of serranid fishes (Perciformes: Teleostei). Zool Stud. 1999;38: 287–294.
- 100.
IBM Corp. IBM SPSS statistics for windows, version 24.0. IBM Corp. 2016.
- 101. Shaw KL. Conflict between nuclear and mitochondrial DNA phylogenies of a recent species radiation: What mtDNA reveals and conceals about modes of speciation in Hawaiian crickets. Proc Natl Acad Sci. 2002;99: 16122–16127. pmid:12451181
- 102. Xiao JH, Wang NX, Li YW, Murphy RW, Wan DG, Niu LM, et al. Molecular approaches to identify cryptic species and polymorphic species within a complex community of fig wasps. PLoS One. 2010;5: pmid:21124735
- 103. Dupuis JR, Roe AD, Sperling FAH. Multi-locus species delimitation in closely related animals and fungi: One marker is not enough. Mol Ecol. 2012;21: 4422–4436. pmid:22891635
- 104. Finnegan AK, Griffiths AM, King RA, Machado-Schiaffino G, Porcher JP, Garcia-Vazquez E, et al. Use of multiple markers demonstrates a cryptic western refugium and postglacial colonisation routes of Atlantic salmon (Salmo salar L.) in northwest Europe. Heredity. 2013;111: 34–43. pmid:23512011
- 105. Fontaneto D, Flot JF, Tang CQ. Guidelines for DNA taxonomy, with a focus on the meiofauna. Mar Biodivers. 2015;45: 433–451.
- 106. Li L, Niu C, Ma R. Rapid temporal succession identified by COI of the rotifer Brachionus calyciflorus Pallas in Xihai Pond, Beijing, China, in relation to ecological traits. J Plankton Res. 2010;32: 951–959.
- 107. Malekzadeh-Viayeh R, Pak-Tarmani R, Rostamkhani N, Fontaneto D. Diversity of the rotifer Brachionus plicatilis species complex (Rotifera: Monogononta) in Iran through integrative taxonomy. Zool J Linn Soc. 2014;170: 233–244.
- 108. Obertegger U, Fontaneto D, Flaim G. Using DNA taxonomy to investigate the ecological determinants of plankton diversity: explaining the occurrence of Synchaeta spp. (Rotifera, Monogononta) in mountain lakes. Freshw Biol. 2012;57: 1545–1553.
- 109. Ojanguren-Affilastro AA, Mattoni CI, Ochoa JA, Ramírez MJ, Ceccarelli FS, Prendini L. Phylogeny, species delimitation and convergence in the South American bothriurid scorpion genus Brachistosternus Pocock 1893: Integrating morphology, nuclear and mitochondrial DNA. Mol Phylogenet Evol. 2016;94:159–170. https://doi.org/10.1016/j.ympev.2015.08.007 pmid:26321226
- 110. Krajíček M, Fott J, Miracle MR, Ventura M, Sommaruga R, Kirschner P, et al. The genus Cyclops (Copepoda, Cyclopoida) in Europe. Zool Scr. 2016;45: 671–682.
- 111. Dömel JS, Melzer RR, Harder AM, Mahon AR, Leese F. Nuclear and mitochondrial gene data support recent radiation within the sea spider species complex Pallenopsis patagonica. Front Ecol Evol. 2017;
- 112. Weisrock DW, Rasoloarison RM, Fiorentino I, Ralison JM, Goodman SM, Kappeler PM, et al. Delimiting species without nuclear monophyly in Madagascar’s mouse lemurs. PLoS One. 2010;5: https://doi.org/10.1371/journal.pone.0009883.
- 113. Birky CW, Fuerst P, Maruyama T. Organelle gene diversity under migration, mutation, and drift: Equilibrium expectations, approach to equilibrium, effects of heteroplasmic cells, and comparison to nuclear genes. Genetics. 1989;121: 613–627. pmid:2714640
- 114. Obertegger U, Cieplinski A, Fontaneto D, Papakostas S. Mitonuclear discordance as a confounding factor in the DNA taxonomy of monogonont rotifers. Zool Scr. 2017;47: 122–132.
- 115. Moore WS. Inferring phylogenies from mtDNA variation: Mitochondrial-gene trees versus nuclear-gene trees. Evolution. 1995;49: 718–726. pmid:28565131
- 116. Eaton TD, Jones SC, Jenkins TM. Species diversity of Puerto Rican Heterotermes (Dictyoptera: Rhinotermitidae) revealed by phylogenetic analyses of two mitochondrial genes. J Insect Sci. 2016;16: 111. http://dx.doi.org/10.1093/jisesa/iew099.
- 117. Cornils A, Held C. Evidence of cryptic and pseudocryptic speciation in the Paracalanus parvus species complex (Crustacea, Copepoda, Calanoida). Front Zool. 2014;11: 19. pmid:24581044
- 118. Cieplinski A, Weisse T, Obertegger U. High diversity in Keratella cochlearis (Rotifera, Monogononta): morphological and genetic evidence. Hydrobiologia. 2018.
- 119. Walsh EJ, Schröder T, Wallace RL, Rico-Martinez R. Cryptic speciation in Lecane bulla (Monogononta: Rotifera) in Chihuahuan Desert waters. Verh. Internat. Verein Limnol. 2009;30: 1046–1050.
- 120. Bilton DT, Freeland JR, Okamura B. Dispersal in freshwater invertebrates. Annu Rev Ecol Syst. 2001; 32: 159–181.
- 121. Boileau MG, Hebert PDN, Schwartz SS. Non-equilibrium gene frequency divergence: persistent founder effects in natural populations. J Evol Biol. 1992;5: 25–39.
- 122. Frisch D, Havel JE, Weider LJ. The invasion history of the exotic freshwater zooplankter Daphnia lumholtzi (Cladocera, Crustacea) in North America: a genetic analysis. Biol Invasions. 2013;15: 817–828.
- 123. Xiang XL, Xi YL, Wen XL, Zhang G, Wang JX, Hu K. Genetic differentiation and phylogeographical structure of the Brachionus calyciflorus complex in eastern China. Mol Ecol. 2011;20: 3027–3044. pmid:21672065
- 124. Derycke S, Backeljau T, Moens T. Dispersal and gene flow in free-living marine nematodes. Front Zool. 2013;10; pmid:23356547
- 125. Fontanillas E, Welch JJ, Thomas JA, Bromham L. The influence of body size and net diversification rate on molecular evolution during the radiation of animal phyla. BMC Evol Biol. 2007;7: 95. pmid:17592650
- 126. Gómez A, Serra M, Carvalho GR, Lunt DH. Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (Rotifera). Evolution. 2002;56: 1431–1444. pmid:12206243
- 127. Mcgovern TM, Hellberg ME. Cryptic species, cryptic endosymbionts, and geographical variation in chemical defences in the bryozoan Bugula neritina. Mol Ecol. 2003;12: 1207–1215. pmid:12694284
- 128. Zhan A, Macisaac H, Melania C. Invasion genetics of the Ciona intestinalis species complex: from regional endemism to global homogeneity. Mol Ecol. 2010;19: 4678–4694. pmid:20875067
- 129. Xavier JR, Rachello-Dolmen PG, Parra-Velandia F, Schönberg CHL, Breeuwer JAJ, van Soest RWM. Molecular evidence of cryptic speciation in the "cosmopolitan" excavating sponge Cliona celata (Porifera, Clionaidae). Mol Phylogenet Evol. 2010;56: 13–20. https://doi.org/10.1016/j.ympev.2010.03.030 pmid:20363344
- 130. Fontaneto D, Giordani I, Melone G, Serra M. Disentangling the morphological stasis in two rotifer species of the Brachionus plicatilis species complex. Hydrobiologia. 2007;583: 297–307.
- 131. Lee CE, Frost BW. Morphological stasis in the Eurytemora affinis species complex (Copepoda: Temoridae). Hydrobiologia. 2002;480: 111–128.
- 132. Lavoué S, Miya M, Arnegard ME, McIntyre PB, Mamonekene V, Nishida M. Remarkable morphological stasis in an extant vertebrate despite tens of millions of years of divergence. Proc R Soc B Biol Sci. 2011;278: 1003–1008. pmid:20880884
- 133. Richards VP, Stanhope MJ, Shivji MS. Island endemism, morphological stasis, and possible cryptic speciation in two coral reef, commensal Leucothoid amphipod species throughout Florida and the Caribbean. Biodivers Conserv. 2012;21: 343–361.
- 134. Salt GW, Sabbadini GF, Commins ML. Trophi morphology relative to food habits in six species of rotifers (Asplanchnidae). Trans Am Microsc Soc. 1997;97: 469–485.
- 135. Wallace RL, Starkweather PL. Clearance rates of sessile rotifers: In situ determinations. Hydrobiologia. 1983;104: 379–383.
- 136. Segers H. Contribution to a revision of Floscularia Cuvier, 1798 (Rotifera: Monogononta): notes on some neotropical taxa. Hydrobiologia. 1997;354: 165–175.
- 137. Gilbert JJ, Walsh EJ. Brachionus calyciflorus is a species complex: Mating behavior and genetic differentiation among four geographically isolated strains. Hydrobiologia. 2005;546: 257–265.
- 138. Schröder T, Walsh EJ. Genetic differentiation, behavioural reproductive isolation and mixis cues in three sibling species of monogonont rotifers. Freshw Biol. 2010;55: 2570–2584. pmid:21116463
- 139. Kim HJ, Iwabuchi M, Sakakura Y, Hagiwara A. Comparison of low temperature adaptation ability in three native and two hybrid strains of the rotifer Brachionus plicatilis species complex. Fish Sci. 2017;83: 65–72.
- 140. Gabaldón C, Fontaneto D, Carmona MJ, Montero-Pau J, Serra M. Ecological differentiation in cryptic rotifer species: what we can learn from the Brachionus plicatilis complex. Hydrobiologia. 2017;796: 7–18.