DNA BARCODING FRESHWATER ROTIFERA OF MEXICO

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1 DNA BARCODING FRESHWATER ROTIFERA OF MEXICO Alma Estrella García Morales Manuel Elías Gutiérrez Zooplankton Laboratory El Colegio de la Frontera Sur, Chetumal, Mexico

2 INTRODUCTION Systematics in Rotifera is far to be accomplished. REASONS: Rotifers are small: microns. There are few taxonomists trained to identify rotifers and cover taxonomic gaps. Intraspecific morphological variation such as high phenotypic plasticity. Interspecific morphological similarity because of morphological stasis. Resulted in: a) undetected cryptic speciation b) species erroneously described c) Synonymies

3 INTRODUCTION Molecular studies with COI Some molecular studies have detected cryptic speciation in few rotifer taxa: Brachionus plicatilis complex, B. calyciflorus, Epiphanes senta, Keratella cochlearis and Lecane bulla. For example in Brachionus plicatilis complex, 14 cryptic species have been found. It seems that cryptic speciation is probably common in rotifers. Molecular techniques can be a tool to understand the nature of genetic differentation and eventually would have an impact on rotifer taxonomy.

4 Our goal is: To test the utility of DNA barcodes by the first time in the identification of a high number of monogonont rotifers from Mexico.

5 STUDY AREA Diverse localities: Baja California Peninsula Central and southeast Mexico

6 COLLECTING METHODS Standardized techniques with zooplankton nets Samples were taken in wetlands, ponds, sinkholes and lakes including littoral and limnetic zones Morphological identification

7 COI SEQUENCES One single rotifer in a PCR tube We used a modified Hot-Shot extraction protocol (Montero-Pau et al. 2008) PCR amplifications according to the standard Barcode of Life protocol (Hajibabaei et al. 2005) 1 rotifer 1 sequence K2P distance model Neighbour-Joining tree

8 RESULTS Sequenced specimens: 461 Sequenced species: 133 In general 69% of the species identified by morphological methods were confirmed by barcodes. 31% remaining morphs were not assigned to a binomial name or could represent possible cryptic species.

9 RESULTS Genetic Distance Comparisons within Species Number of comparisons 1151 Minimum (%) 0 Distance mean (%) 0.73 Maximum (%) S.E. (%) Genera Families Orders

10 HIGHLIGHTING POSSIBLE CRYPTIC SPECIES Lecane bulla L. cornuta L. curvicornis L. hastata L. luna L. lunaris Brachionus calyciflorus B. quadridentatus f. brevispinus B. quadridentatus f. cluniorbicularis Mytilina ventralis var. macracantha Keratella cochlearis K. tropica Platyias quadricornis Testudinella patina Ascomorpha ovalis

11 Lecane bulla (Gosse, 1851) Eurytopic and common taxon with high intraspecific variability (Segers 1995). In 22 populations from the Chihuahuan desert 3 genetic clusters were found (Walsh et al. 2009). We analysed 18 populations and Barcodes revealed 8 genetic clusters, which averaged 5% divergence.

12 Lecane cornuta (O. F. Müller, 1786) This cosmopolitan species live between aquatic vegetation. L. cornuta also exhibits intraspecific morphological variation (Segers 1995) Barcodes separated to L. cornuta in 3 genetic groups showing a mean divergence of 6.3%.

13 Mytilina ventralis var. macracantha macracantha is the long-spined variety of the M. ventralis species. This taxon inhabits in littoral zones, mainly in the aquatic vegetation. The shape and size of lorica and spines in this species is variable Barcodes detected 4 genetic clusters (Veracruz, Campeche, Quintana Roo, State of Mex.). Clusters averaged 6.8% divergence.

14 TAXONOMIC CONTROVERSIES Genus Brachionus This warm-water genus is the most diverse in the Brachionidae family. The taxonomy of the Brachionus species is highly controversial due to their extensive phenotypic variation, which has led to many forms have been described. But it is difficult to know if these forms are just morphological variations of a single species or whether they represent different species. B. quadridentatus f. brevispinus f. rhenanus f. cluniorbicularis

15 Brachionus quadridentatus (Hermann, 1783) 1 B. quadridentatus f. brevispinus B. quadridentatus f. cluniorbicularis The mean genetic distance between these two forms was 7.5%. These results indicate that the forms are different species.

16 Brachionus havanaensis Rousselet 1911 The specimens examined showed some morphological differences in the spine length and body size. We analyzed 9 populations which showed relatively low sequence divergences (mean 1.4%).

17 Brachionus havanaensis Rousselet 1911 Despite the morphological variations presented in this species, the barcode congruence observed among the populations confirms their high variability in response to environmental pressures.

18 Brachionus plicatilis complex From 14 possible species, only three have been formally described. We found three geographically distant populations of the B. plicatilis complex with low divergence. Blast searches confirmed our individuals as the Brachionus sp. Almenara isolate Indianrocks1 from USA with 99% similarity, sequenced by the first time from a strain collected in Spain. The similar genetic variation between our specimens, the USA and Spain populations suggests that colonization events happened relatively recently.

19 CONCLUSIONS Barcodes were successful in discriminating 69% of the species in our study. The present work provides an example how barcodes can be a complementary tool to support species identification and flagging species that need more analysis. To fully resolve the relationships among cryptic species, additional morphological, ecological, biogeographical evidence and reciprocal mating experiments are needed. It is necessary to study more ample geographic areas in order to elucidate the full extent of genetic differentiation of rotifer taxa.

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