Biodiversity, taxonomy and systematics of new world freshwater leeches (Annelida: Hirudinea) with particular emphasis on glossiphoniid leeches and their bacterial endosymbionts

The phylum Annelida Lamarck, 1809 includes segmented worms such as leeches, earthworms, lugworms, sandworms and clamworms that inhabit almost all possible environments and places of the world. Leeches (Class Hirudinea) represents only one group of around 680 species out of the approximately 16,500 described species of Annelida. The class Hirudinea has been divided in two groups based on their mouthparts. The order Rhynchobdellida, a paraphyletic assemblage, includes species with a large and eversible proboscis and the order Arhynchobdellida that includes species with a muscular pharynx with or without jaws. Both orders include organisms specialized to feed on vertebrate blood. This study includes the description of eight species of leeches new to science that belong to three families (Glossiphoniidae, Macrobdellidae and Praobdellidae). The phylogenetic relationships of species of three families (Glossiphoniidae, Macrobdellidae and Praobdellidae) and one suborder (Erpobdelliformes) were investigated using molecular and morphological data and a suite of phylogenetic methods (Parsimony, Maximum Likelihood and Bayesian Inference). The description of the new species Tyrannobdella rex (Praobdellidae) and Oxyptychus bora (Macrobdellidae) are discussed in the context of their placement in phylogenies. The phylogenetic study of Erpobdelliformes includes the comparison of alternative classification schemes. Based on the results, the phylogenetic position of the terrestrial and macrophagous Orobdella octonaria (Gastrostomobdellidae) within the Erpobdelliformes is established for the first time. The phylogenetic relationships of the proboscis-bearing species of the genera Haementeria, Helobdella and Placobdella were investigated using a combination of nuclear and mitochondrial markers and Parsimony and Bayesian Inference methods. In addition to the monophyly of Haementeria, Helobdella and Placobdella, the 3 genera formed a monophyletic group notwithstanding their different feeding preferences. The correlation with phylogeny and some morphological traits is shown. These include, eyespot morphology, annulation patterns, shape of the ovisacs, sensory organs on the dorsal surface and presence of bacteriomes. Species of Haementeria and Placobdella have specialized organs called bacteriomes associated with their salivary complex that harbor symbiotic proteobacteria. Using DNA bacterial sequences (16S rRNA), the exclusive association of Haementeria spp. with gammaproteobacteria and Placobdella spp. with alphaproteobacteria is shown. Using pyrosequencing technology, the nucleotide sequences of a DNA sample extracted from the bacteriomes of Placobdella parasitica were analyzed. A total of 1,053,345 DNA fragments were obtained and assembled. Leech and symbiont DNA fragments were separated using Blast tools and 50 bacterial and Helobdella robusta genomes for reference. Finally, the so-called DNA barcoding protocol is discussed and some recommendations were given to increase the information content of the database (Bold system). In addition, DNA barcoding protocol was used to estimate the diversity of species of Helobdella from Mexico.