Post-Miocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella D. S. Zarlenga†‡§, B. M. Rosenthal‡, G. La Rosa¶, E. Pozio¶, and E. P. Hoberg‡ †Bovine Functional Genomics Laboratory and ‡Animal Parasitic Diseases Laboratory and U.S. National Parasite Collection, U.S. Department of Agriculture, Beltsville, MD 20705; and ¶Department of Infectious, Parasitic, and Immunomediated Diseases, Istituto Superiore di Sanita`, 00161 Rome, Italy Communicated by William C. Campbell, Drew University, Madison, NJ, March 27, 2006 (received for review December 16, 2005) Parasitic nematodes of the genus Trichinella cause significant diversity in host–parasite associations, suggesting that parasites food-borne illness and occupy a unique evolutionary position at of vertebrates arose independent of one another during the long the base of the phylum Nematoda, unlike the free-living nematode evolutionary history for nematodes (3). Thus, understanding the Caenorhabditis elegans. Although the forthcoming genome se- origins and persistence of vertebrate parasitism requires phylo- quence of Trichinella spiralis can provide invaluable comparative genetic and historic information from a broad spectrum of taxa. information about nematode biology, a basic framework for un- Among the Trichocephalida, parasitism may have arisen as early derstanding the history of the genus Trichinella is needed to as the Paleozoic in the ancestor of the Trichinellidae and the maximize its utility. We therefore developed the first robust and Trichuridae; however, temporal origins of extant species of comprehensive analysis of the phylogeny and biogeographic his- Trichinella remain unexplored. tory of Trichinella using the variation in three genes (nuclear The genus Trichinella comprises a monophyletic lineage in the small-subunit rDNA, and second internal transcribed spacer, mito- Trichinellidae, the putative sister to the Trichuridae (Capillari- chondrial large-subunit rDNA, and cytochrome oxidase I DNA) from inae, Trichurinae, and Trichosomoidinae). The superfamily all 11 recognized taxa. We conclude that (i) although Trichinellidae Trichinelloidea to which Trichinella belongs is phylogenetically may have diverged from their closest extant relatives during the diagnosed by the stichosome, a region of the glandular esoph- Paleozoic, all contemporary species of Trichinella diversified within agus, and the bacillary bands, an assembly of structural charac- the last 20 million years through geographic colonization and ters otherwise unknown among the nematodes. Trichinellidae is pervasive host switching among foraging guilds of obligate car- further diagnosed by a unique life cycle where worms undergo nivores; (ii) mammalian carnivores disseminated encapsulated complete development within a single vertebrate host (autohex- forms from Eurasia to Africa during the late Miocene and Pliocene, eroxeny) and by first-stage larvae, which localize within skeletal and to the Nearctic across the Bering Land Bridge during the muscle cells as infectious organisms. Although eating raw or Pliocene and Pleistocene, when crown species ultimately diversi- undercooked pork infected with T. spiralis accounts for most fied; (iii) the greatest risk to human health is posed by those species human cases of trichinellosis, zoonotic transmission also occurs retaining an ancestral capacity to parasitize a wide range of hosts; from species that circulate among wildlife. Eleven distinct taxa and (iv) early hominids may have first acquired Trichinella on the are now recognized in mammals, birds, crocodilians, and sauri- African savannah several million years before swine domestication ans (5), characterized by either a thick (encapsulated) or a very as their diets shifted from herbivory to facultative carnivory. thin (nonencapsulated) collagen membrane enveloping the par- asite-infected muscle cell. biogeography ͉ mitochondrial DNA ͉ phylogeny ͉ ribosomal DNA Despite extensive development of biochemical and genetic methods to distinguish taxa (6), evolutionary relationships xceptional biological diversity among nematodes is exempli- among genotypes of extant Trichinella remain enigmatic. Delin- Efied by certain attributes of the parasite Trichinella spiralis. eating the phylogeny and biogeography of Trichinella will sub- Organization of its mitochondrial genome more closely resem- stantially increase the comparative value of its genome sequence bles that of coelomate metazoans than that of its presumed and help elucidate the determinants of zoonotic risk among closest relatives, the secernentean nematodes (1). In addition, T. species. On a broader scale, this information will provide us an spiralis (Dorylaimia) shares a similar proportion (45%) of its opportunity to investigate ecological and evolutionary forces ESTs with the nematode Caenorhabditis elegans (Rhabditina) as that promote diversification in complex host–parasite systems. it does with the fruit fly Drosophila melanogaster (Arthropoda: Three central questions fueled our curiosity about the evolu- Drosophilidae) (2). Thus, many ESTs common to T. spiralis and tion of Trichinella.(i) Can prolonged coevolution with distinct C. elegans are not necessarily specific to nematodes but may be tetrapod groups explain why encapsulated species of Trichinella conserved among diverse taxonomic groups of invertebrates. C. are restricted to eutherian mammals and nonencapsulated spe- elegans is often thought of as a prototypical nematode because cies also infect saurian, crocodilian, avian, eutherian, and met- of its acceptance as a model for studying biological processes; atherian hosts (5, 7), or have more recent ecological and however, genomic variation among nematodes is extensive and biogeographic events influenced current host associations? (ii) commensurate with their phylogenetic and ecological diversity. Why are most species and genotypes with broad host ranges Therefore, the forthcoming genome sequence of T. spiralis will contribute substantially to our understanding of nematode bi- Conflict of interest statement: No conflicts declared. ology and the origins of parasitism. Abbreviations: SSU, small-subunit; LSU, large-subunit; ITS-2, second internal transcribed In 1998, Blaxter et al. (3) used genetic data to delineate the spacer; ML, maximum likelihood; MY, million years; COI, cytochrome oxidase I. phylum Nematoda into supertaxa consisting of five clades. Data deposition: The sequences reported in this paper have been deposited in the GenBank Trichinella, a parasite of vertebrates, occupies a basal lineage database (accession nos. AY851256–AY851277 and DQ007890–DQ007900). (clade I) consisting of free-living Mononchida, plant parasitic §To whom correspondence should be addressed at: U.S. Department of Agriculture, Agri- Dorylaimida, and entomophagous Mermithida, with which it cultural Research Service, Bovine Functional Genomics Laboratory, B1180 Beltsville Agri- shares features of early embryogenesis (4) and small-subunit cultural Research Center East, Beltsville, MD 20705. E-mail: [email protected]. (SSU) rDNA sequences (3). Other clades show similar levels of © 2006 by The National Academy of Sciences of the USA 7354–7359 ͉ PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602466103 Downloaded by guest on September 28, 2021 Table 1. Characteristics of sequence alignments and their evolutionary models No. of bases† Base frequencies Clock§ Model Data set* Total Var. Inf. type‡ C:T͞Tv G:A͞Tv A C G I ⌫ shape ⌬InL df Ͼ P SSU 1,929 717 417 TrNef⌫ 2.80 2.06 0.250 0.250 0.250 0 0.408 20.3 14 Ͼ 0.12 11mit 1,193 276 194 HKY ⌫ I 15.60 15.60 0.299 0.170 0.174 0.49 0.492 9.84 9 Ͼ 0.35 3gene 1,273 204 78 HKY ⌫ 3.21 3.21 0.277 0.165 0.200 0 0.156 1.86 6 Ͼ 0.93 *Data sets are as follows: SSU, 10 of 11 Trichinella genotypes including outgroup taxa Trichuris muris, Trichuris suis, Adnoncholaimus sp., Pirapulus caudatus, Mormis nigrescens, and Mylonchulus arenicolu; 11mit, mitochondrial LSU rDNA and COI from all 11 Trichinella taxa; 3gene, mitochondrial LSU rDNA, COI, and ITS-2 from all eight encapsulated species and genotypes of Trichinella. †Total, variable (Var.), and informative (Inf.) number of bases under the parsimony criterion. ‡TrNef, a model consisting of a single transversion rate but two distinct transition rates. HKY models correspond to that of Hasegawa et al. (8) in assuming unequal base frequencies and rates of transition and transversion substitutions. ⌫, variation in the substitution across nucleotide sites modeled as a gamma distribution with an estimated shape parameter; I, the estimated proportion of invariant sites; Tv, transversion. §Difference in log likelihood (⌬InL) trees evaluated with and without the enforcement of a molecular clock. df ϭ (number of taxa) Ϫ2. P represents statistical significance in the reduction of likelihood owing to enforcement of a molecular clock. restricted to well defined geographical localities? (iii) Did hu- among all species. These values are substantially less than the mans become infected with Trichinella in the Neolithic before ML distance (0.2) separating Trichuris muris from Trichuris suis. domesticating pigs, or did adaptation after pig domestication If the locus evolved in each lineage at Ϸ0.4 ϫ 10Ϫ9 per site per cause certain species of Trichinella to threaten human health? To year (4% over 100 MY), which typifies many metazoan phyla (9), explore these and related questions, we generated the first robust then