Molecular phylogeny of the genus Margotrema (Digenea: Allocreadiidae), parasitic flatworms of goodeid freshwater fishes across central Mexico: species boundaries, host-specificity, and geographical congruence ANDRÉS MARTÍNEZ-AQUINO 1,2 *, FADIA SARA CECCARELLI 3 and GERARDO PÉREZ-PONCE DE LEÓN 1 1 Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-153, Ciudad Universitaria, México, D.F., México 2 Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Av. Ciudad Universitaria 3000, C.P. 04360, Coyoacán, Distrito Federal, México 3 Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-153, Ciudad Universitaria, México, D.F., México Received 8 November 2012; revised 30 January 2013; accepted for publication 1 February 2013 We explored the genetic variation at the intraspecific level of two putative species of Margotrema, M. bravoae and M. guillerminae, to establish the species boundaries. Sequences of a mitochondrial (COI) and nuclear (ITS1) gene were obtained for 125 specimens distributed in 15 localities from seven hydrological systems. An alignment of 750 and 831 bp including gaps of COI and ITS1, respectively, was assembled. We analysed the gene fragments separately as well as together by using Bayesian inference for phylogenetic reconstruction. Based on the phylogenetic analyses, an ultrametric tree was built for each gene, and a general mixed Yule-coalescent model for species delimitation was carried out. A multispecies coalescent analysis was performed in *BEAST using both molecular markers. The results show four independent evolutionary lineages that we interpret as two valid species of Margotrema; the first is represented by an independent lineage, and the second is composed of three lineages. Because the species M. bravoae and M. guillerminae nested together within these three lineages, the validity of M. guillerminae as an independent species is questioned, and it is proposed to represent a junior synonym of M. bravoae. Each lineage shows a congruent geographical pattern with respect to the hydrological system where they occur. Additionally, each lineage shows congruence with respect to the hosts they parasitize, either at the species or at higher taxonomical levels (tribe). The parasite evolutionary history is congruent with the evolutionary and biogeographical history of their hosts. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 168, 1–16. doi: 10.1111/zoj.12027 ADDITIONAL KEYWORDS: COI – GMYC – goodeinae – ITS1 – species tree. INTRODUCTION Our understanding of the evolution of parasitic organ- isms, as well as the complex and intricate association they establish with their hosts, has greatly improved with the use of molecular tools and a range of ana- lytical methods that are available for analysing these data sets. Advancements in methodological tools has resulted in establishing more robust species delimitation criteria for parasitic organisms and understanding the influence of host specificity and *Corresponding author. E-mail: maandres@ibiologia.unam.mx Zoological Journal of the Linnean Society, 2013, 168, 1–16. With 5 figures © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 168, 1–16 1