Date of Award


Degree Name

Doctor of Philosophy


Biological Sciences


Elizabeth J. Walsh


Speciation is a continuous and adaptive process by which lineages are diverged into multiple groups, and species are the product of this process. Taxonomy is the study of relationship between organisms, classifying and naming them, and one of the taxonomical challenges is delimiting species boundaries. Species delimitation can be controversial because biologists do not agree on species concepts and approaches for defining species boundaries. One of the well-known species concepts is the Biological Species Concept that requires studying reproductive barriers among populations. Investigating strength of reproductive isolation among populations is not always practical in the wild. Therefore, many biologists have used morphological traits as an indicator of reproductive isolation and for delineating species. Yet, morphological methods are not fully effective in defining species boundaries and detecting species diversity, since some species are morphologically identical. Molecular analyses have contributed to species delimitation for morphologically indistinguishable groups (cryptic species). Species that are delimited based on molecular methods have been further tested using multiple complementary approaches (integrative taxonomy) such as ecological, behavioral and morphological differentiation especially for groups such as microorganisms that show high morphological uniformity.

Rotifers, similar to other microorganisms, have drought-resistance propagules that are efficient for long distance dispersal. Therefore, they are assumed to have high rates of gene flow among habitats even across large geographic scales. As a result of high population connectivity, little genetic variation in population structure within rotifer morphospecies is expected. Moreover, rotifers do not have a lot of recognizable morphological characteristics and there has not been enough effort to resolve the taxonomical controversies resulted from morphological plasticity and cryptic species; morphologically similar species (cryptic species) are often not distinguished. However, high genetic structure has been reported among populations of many rotifer morphospecies suggesting they are species complexes with multiple cryptic species. Thus, rotifers are a good model organism for the application of molecular methods for species delimitation and to test the DNA based species boundaries using an integrative taxonomy. Integrating multiple approaches has been successfully used to delimit species boundaries in some rotifer species complexes such as Epiphanes senta and Brachionus calicyflorus.

In Chapters 1 and 2, I used COI gene and ITS region sequences to study genetic structure and to delimit cryptic species in a littoral rotifer morphospecies, Euchlanis dilatata (62 populations), and four sessile morphospecies (Limnias melicerta [29 populations]; L. ceratophylli [20 populations]; Collotheca campanulata [19 populations]; C. ornata [45 populations]). Using Bayesian species delimitation (BSD), I found seven putative cryptic species for E. dilatata based on the ITS region sequence analysis. Based on COI gene sequences analyzed by BSD, nine putative cryptic species within L. melicerta, four putative cryptic species within L. ceratophylli, seven putative cryptic species for C. campanulata and eight putative cryptic species for C. ornata were detected. The relationship between genetic and geographic distance was weak or lacking within the examined morphospecies. Moreover, geographic distributions of cryptic species varied from occurring in a single locality, broadly, or even overlapping suggesting that they may differ in their capabilities to disperse, colonize, and persist in new habitats. Geometric and morphometric analyses did not show significant variation in trophi (rotifer's jaws) shape and size among cryptic species of L. melicerta and L. ceratophylli. The lack of morphological variation can be a case of morphological stasis 1) through stabilizing selection because of niche conservationism, or 2) a result of speciation mediated by ecological and/or mating signals differentiation without morphological changes.

In Chapter 3, to test the species boundaries defined by a molecular approach, I investigated reproductive isolation, variation in trophi morphology and life history characteristics among cryptic species of E. dilatata. Mating success rate between each cryptic species was 0-1.1%, which was lower than that of positive controls (intra-clonal: 15.6-43.9%; Chi-Square= 15.3-52.2, p<0.001). SEM trophi images representing the seven cryptic species of E. dilatata were used for morphometric analyses. Using Discriminant analysis, 64% of individuals were correctly assigned to cryptic species (Chi-Square= 78, p< 0.001); trophi morphology cannot be used to distinguish E. dilatata cryptic species except for cryptic species A. To investigate life history characteristics of cryptic species, four treatments were used: (1) 20Ë?C, 180 µS/cm, (2) 20Ë?C, 1800 µS/cm, (3) 27Ë?C, 180 µS/cm, and (4) 27Ë?C, 1800 µS/cm. The interaction between temperature and conductivity had significant effects on generation time, net reproductive rate, and the intrinsic rate of population increase in some of the cryptic species (p=0.03). All cryptic species had higher survivorship and fecundity under temperature 27Ë?C while showing variation in response to water conductivity. My findings showed cryptic species of E. dilatata are reproductively isolated and they show differentiation in life history characteristics although except for one cryptic species they cannot be distinguished based on morphology. Because I provided support for the DNA taxonomy species boundaries by finding they are reproductively isolated and ecologically differentiated, I described four cryptic species of E. dilatata as new species. A specimen from cryptic species A was selected as neotype for Euchlanis dilatata because this species showed the widest geographic distribution in USA and Mexico. In Chapter 4, I used ddRAD-Seq to investigate the molecular basis of ecological adaptation and to gauge adaptive genetic variation between two cryptic species of E. dilatata. I obtained 107 loci that were present in at least 40% of individuals for seven populations representing two proposed cryptic species. FST values ranged from 0-0.95 indicating there was high genetic differentiation between them. Most populations from different cryptic species showed high genetic divergence. One exception was the FST value of 0 between Cattle Tank, NM (cryptic species D) and Triangle Pond, AZ (cryptic species C). The genetic similarity between these two populations could be a result of admixture, or inefficiency of obtained loci in representing accurate amount of genetic divergence among populations of E. dilatata. There was no significant relationship between genetic variation at those loci and geographic distance among populations. However, in Discriminant Analysis of Principle Components, populations that were collected from similar habitats were grouped together. This indicates there could be a relationship between genetic variation and habitat features. To provide support for these results, additional samples should be included and only loci with coverage across at least 80% of individuals should be retained for downstream analysis.

Finally, in my research on cryptic species of rotifers, I was able to show that isolation by distance was not strongly related to the observed genetic variation. On the other hand, as it was shown by ddRAD-Seq analyses of E. dilatata, genetic divergence may be related to ecological adaptation. However, because of the low number of loci and their low coverage I was not able to find genes that could be related to ecological adaptation. Further studies focused on genomic regions with adaptive functions among rotifer cryptic species will obtain a better understanding of the ecological speciation mechanisms in rotifers.

I studied cryptic diversity within Euchlanis dilatata and for the first time, for four sessile morphospecies. I used DNA-based taxonomy to delimit species boundaries for morphologically similar lineages within five rotifer morphospecies. Integrative taxonomy has been suggested for species delimitation in especially in groups with limited morphological characteristics such as microorganisms. Here, I used integrative approach for more reliable species delimitation within Euchlanis dilatata and I found that those cryptic species are reproductively isolated and ecologically differentiated. On the other hand, I showed that genetic diversity within each morphospecies has weak correlation with geographic isolation. This may indicate that speciation in rotifers is not necessarily caused by geographic isolation. On the other hand, genetic variation among cryptic species is potentially associated to differentiation in ecological adaptation. Therefore, cryptic species of rotifers are likely to show variation in their adaptive range resulting in genetic variation and reproductive isolation among them. This mode of speciation is not necessarily accompanied by morphological divergence.




Received from ProQuest

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331 pages

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Azar Kordbacheh

Available for download on Thursday, June 04, 2020