Revealing the Secret Lives of Cryptic Species: Examining the Phylogenetic Relationships of Echinostome Parasites in North America
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ARTICLE IN PRESS Molecular Phylogenetics and Evolution xxx (2010) xxx–xxx Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Revealing the secret lives of cryptic species: Examining the phylogenetic relationships of echinostome parasites in North America Jillian T. Detwiler *, David H. Bos, Dennis J. Minchella Purdue University, Biological Sciences, Lilly Hall, 915 W State St, West Lafayette, IN 47907, USA article info abstract Article history: The recognition of cryptic parasite species has implications for evolutionary and population-based stud- Received 10 August 2009 ies of wildlife and human disease. Echinostome trematodes are a widely distributed, species-rich group of Revised 3 January 2010 internal parasites that infect a wide array of hosts and are agents of disease in amphibians, mammals, and Accepted 5 January 2010 birds. We utilize genetic markers to understand patterns of morphology, host use, and geographic distri- Available online xxxx bution among several species groups. Parasites from >150 infected host snails (Lymnaea elodes, Helisoma trivolvis and Biomphalaria glabrata) were sequenced at two mitochondrial genes (ND1 and CO1) and one Keywords: nuclear gene (ITS) to determine whether cryptic species were present at five sites in North and South Cryptic species America. Phylogenetic and network analysis demonstrated the presence of five cryptic Echinostoma lin- Echinostomes Host specificity eages, one Hypoderaeum lineage, and three Echinoparyphium lineages. Cryptic life history patterns were Molecular phylogeny observed in two species groups, Echinostoma revolutum and Echinostoma robustum, which utilized both Parasites lymnaied and planorbid snail species as first intermediate hosts. Molecular evidence confirms that two Trematodes species, E. revolutum and E. robustum, have cosmopolitan distributions while other species, E. trivolvis Mitochondrial markers and Echinoparyphium spp., may be more geographically limited. The intra and interspecific variation Nuclear markers detected in our study provides a genetic basis for seven species groups of echinostomes which will help accurately identify agents of disease as well as reveal cryptic aspects of trematode biology. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction 2009), though in the case of parasites with complex life cycles, it is often less clear which host may have the most influence on par- The existence of cryptic species occurs because of morphologi- asite speciation. Several mechanisms of host-mediated structuring cal similarity and appears to be wide-spread among metazoan taxa include host feeding strategies (Jousson et al., 2000; Criscione et al., and geographical regions (Pfenninger and Schwenk, 2007). Follow- 2002) and host immune systems (Štefka et al., 2009). The recogni- ing Bickford et al. (2007), we define cryptic species as two or more tion of cryptic parasite species allows for an accurate assessment of distinct lineages that are classified as a single species, owing to the parasite diversity, a biological indicator of species-richness in the lack of conspicuous morphological differences. Crypsis among spe- free-living host community (Hudson et al., 2006). By measuring cies is often discovered through molecular genetic, behavioral, or the changing abundances of parasite populations over time, we ecological studies of diversity. When revealed, cryptic species com- can predict when there is an increasing risk of disease (Johnson plexes clarify and modify our views of biodiversity and biogeogra- and McKenzie, 2009). Identifying cryptic species among parasites phy, life history, evolutionary theory, and ecological interactions reveals the unrecognized diversity in the sources and mechanisms (Bickford et al., 2007). For instance, elucidation of crypsis may shed of human and wildlife diseases. Failing to recognize cryptic species light on species diversity, the process of speciation, and the leads to inaccurate assessments of parasite prevalence and inten- dynamics of populations and demes. sity, underestimates of multi-species infections, and lost opportu- Parasitic species are a large and diverse group of organisms and nities to study parasite–parasite interactions. Ultimately, resolving are no exception to the commonality of cryptic speciation (Jousson relationships among cryptic parasite species will help us under- et al., 2000; Steinauer et al., 2007; Miura et al., 2005; Vilas et al., stand the dynamics of parasite/disease dispersal, mechanisms 2005). While mechanisms of speciation are similar between free- underlying parasite speciation, and other factors involved in para- living and parasitic species, parasites differ in that speciation pat- site epidemiology. Such understanding serves as the basis for inter- terns may be directly associated with their hosts (Malenke et al., vention, treatment, and prevention of disease caused by parasites (Bickford et al., 2007; Putaporntip et al., 2009). Echinostome trematodes (Echinostomatidae) are a diverse and * Corresponding author. Fax: +1 765 494 0876. E-mail addresses: [email protected] (J.T. Detwiler), [email protected] (D.H. ubiquitous group of parasites that are commonly used as a model Bos), [email protected] (D.J. Minchella). system in laboratory and field investigations. In addition to infect- 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.01.004 Please cite this article in press as: Detwiler, J.T., et al. Revealing the secret lives of cryptic species: Examining the phylogenetic relationships of echinostome parasites in North America. Mol. Phylogenet. Evol. (2010), doi:10.1016/j.ympev.2010.01.004 ARTICLE IN PRESS 2 J.T. Detwiler et al. / Molecular Phylogenetics and Evolution xxx (2010) xxx–xxx ing numerous avian and mammalian species, echinostomes are host specificity to the first intermediate host (Kostadinova and Gib- increasingly associated with disease in amphibians and may con- son, 2000). Second, morphological data consisting of the number of tribute to their decline (Echinostoma revolutum, Holland et al., spines surrounding the mouth was assessed with voucher speci- 2007; Echinostoma trivolvis, Schotthoefer et al., 2003), especially mens. The number of spines found on adult and larval stages is one in urban environments (Skelly et al., 2006). Establishing spatial of the most common features used to characterize particular species scale of parasite distributions and host use is important in the face groups (Fried, 2001; Fried and Graczyk, 2004). Third, genetic se- of emerging diseases, yet morphological similarity among species quences were used to examine phylogenetic relationships among may mask differences in small-scale spatial distributions of para- taxa for three genes. A better understanding of inter- and intraspe- sites (Leung et al., 2009) and in host specificity (Detwiler and cific variation for echinostomes was determined by comparing ge- Minchella, 2009). Echinostomes are prone to misidentification netic distances and generating haplotype networks. due to similar morphology and the lack of isolates in molecular databases (Kostadinova et al., 2003). For instance, although echi- 2.1. Sampling nostome species can be distinguished by the number of spines on the head collar of the larval and adults stages, some species We collected samples from five field sites in North and South have the same number of spines. More comprehensive sampling America (Table 1) including Indiana, Minnesota, Wisconsin and is needed to establish a better idea of the underlying geographical Brazil. The most extensive sampling occurred from 2005–2008 at and genetic variation within and among lineages. two Indiana sites where repeated snail intermediate host sampling Echinostomes differ from other platyhelminth parasites in sev- was conducted for snail intermediate hosts. A limited number of eral ways. First, echinostomes are species-rich, for example, approx- field-infected hosts were collected from Minnesota (4 snails), Wis- imately 60 and 120 nominal species are described from just 2 of the consin (1 muskrat) and Brazil (1 snail). In addition, we received a 91 recognized echinostome genera (Kostadinova and Gibson, 2000). tissue sample originally collected in the USA that was previously Yet, E. revolutum and E. trivolvis are the only two species from North sequenced at two rRNA genes for a trematode phylogeny (Olson America that appear in a phylogenetic framework (Sorensen et al., et al., 2003). 1998; Olson et al., 2003). Including additional samples from North Field-collected snails were transported to the laboratory and and South America may clarify the phylogenetic relationships of larval parasites were first assigned to species based on host speci- these echinostome species to those from other geographic regions ficity and morphology as described in Detwiler and Minchella around the world. Second, species generally have cosmopolitan dis- (2009). Briefly, cercarial shedding was induced in field hosts, and tributions in which a single species is reported from Europe, Africa, parasites were examined for the presence/absence of collar spines Asia and Australia (Kostadinova et al., 2003). Third, members of this and tail flaps. To obtain adult specimens for spine counts, cercariae parasite group have a broad host range, especially in second inter- from field-infected first intermediate hosts were exposed to labo- mediate and definitive hosts (Kanev et al., 2000). Therefore, they ratory-raised