Molecular systematics of the avian schistosome genus Trichobilharzia (Trematoda: Schistosomatidae) in North America

Abstract

Trichobilharzia is a genus of threadlike schistosomes with a cosmopolitan distribution in birds. Species of Trichobilharzia achieve notoriety as major etiological agents of cercarial dermatitis, or swimmer's itch. There are 40 species described in the literature, for which the majority lacks molecular sequence information. To better understand the phylogenetic relationships, diversity, species boundaries, host use, and geographic distribution of this genus better, we surveyed 378 birds and over 10,000 snails from North America. The phylogenetic analysis was based on nuclear 18S, 28S rDNA, internal transcribed spacer region and mitochondrial cytochrome oxidase I sequence data. Specimens were recovered that could be related to 6 of the 14 described species of Trichobilharzia from North America (Trichobilharzia physellae, Trichobilharzia querquedulae, Trichobilharzia szidati, Trichobilharzia stagnicolae, Trichobilharzia franki, and Trichobilharzia brantae). An additional 5 lineages were found that could not be related directly to previously described species. Trichobilharzia brantae, transmitted by Gyraulus parvus, grouped outside the clade containing the recognized species of Trichobilharzia. A subgroup of the Trichobilharzia clade designated Clade Q was comprised of closely related species whose adults and eggs are similar, yet the European species use lymnaeids whereas the North American species use physids as snail hosts. This molecular phylogeny provides a useful framework (1) to facilitate identification of worms, including those involved in dermatitis outbreaks; (2) to test hypotheses about the evolution, diversification, host-parasite interactions and character evolution of Trichobilharzia; and (3) to guide future taxonomic revision of Trichobilharzia.

FIGURE 1

Collection localities. Refer to Table III for more details. Localities are as follows: 1 Canada: Manitoba, Churchill 58.7541 N; 93.8066 W, July/August 2007; 2 Colorado: El Paso Co. 38.827 N; 104.804 W, June 2007; 3 New Mexico: Bernalillo Co. 35.1305 N; 106.6822 W, July 2002; 4 New Mexico: Sandoval Co. 35.8485 N; 106.4907 W, July 2006; 5 Pennsylvania: Erie Co. 42.1703 N; 80.0868 W, November 2004; 6 New Mexico: Chavez Co. 33.45 N; 104.4 W, April 2005, March 2006; 7 Alaska: North Slope Borough 68.9820 N; 148.8318 W, June 2005; 8 Nevada: Churchill Co. 39.9 N; 118.817W, November 2005; 9 Michigan: Cheboygan Co. 45.581 N; 84.697 W, July, 1999, August 2005; 10 Florida; 11 Louisiana, Cameron Parish 26.661 N; 92.688 W, November 2003; 12 California: Imperial Co. 33.2988 N; 115.5875 W, November 2004; 13 New Mexico: Socorro Co. 33.7131 N 106.9579 W, April 2004; 14 Alaska: Yukon-Koyukuk Borough 65.665 N; 149.098 W, May 2005; 15 Nebraska: Nemaha Co. 40.467 N; 95.7 W, November 2004; 16 Montana: Big Fork Lake Co. 47.483 N; 114.217 W, 1999; 17 New Mexico: Taos Co. 36.8467 N; 105.3794 W, June 2004; 18 Minnesota: Itasca Co. 47.510 N; 94.185 W, July 2008; 19 Michigan: Luce Co. 46.667 N; 85.733 W, July 1999; 20 New Mexico: Sierra Co. 32.9071 N; 107.3116 W, February 2005; 21 Pennsylvania: Crawford Co. 41.575 N; 80.212 W, November 2004.

FIGURE 2

Maximum likelihood tree based on 18S–28S sequences. The schistosomatids are enclosed in the box, and the BTGD clade delimited by shading. Samples in bold are those collected from this study. Within the BTGD clade, individual specimens of Trichobilharzia were collapsed and are labeled only by the taxon name. Node support is indicated by MP and ME bootstrap values and Bayesian posterior probabilities (PP), respectively. The “*” indicates MP and ME bootstrap values of >90 and PP of 100. The ‘-’ indicates no significant node support. Branch support is designated only for the major clades.

FIGURE 3

Maximum likelihood tree based on 18S-28S-cox1-ITS sequences. Trichobilharzia is highlighted. Samples in bold are those collected from this study. Node support is indicated by MP and ME bootstrap values and Bayesian PP, respectively. The ‘*’indicates MP and ME bootstrap values of >90 and PP of 100. The ‘-’ indicates no significant node support. Image is of Trichobilharzia physellae (USNPC# 079636).

FIGURE 4

Maximum likelihood tree based on cox1 sequences. For the Trichobilharzia querquedulae clade only some of the worms sequenced are represented as many differed by only one base pair. The “*” indicates node support of >95% bootstrap for MP and ME and >98 Bayesian PP. The ‘-’ indicates no significant node support. Outgroup species of Schistosoma were collapsed. For convenience, the following taxa were trimmed from the tree, but were fully supported in the clade: W137blteLA, W156blteNM, W148.1citeNM, W148.2citeNM, W155.3citeNM, W158noshNM, W162noshNM, W183noshCA, SDS1006noshNE, E45blteFL (Table III). The same was done for T. physellae, except in one case there were identical haplotypes: TpB = W171lescPA, W193lescNM, W255buheNM, W263PhysaNM. Otherwise, the following with only 1–2 bp differences were removed W211olsqAK, W193lescNM, W236PhysaMI, W230.1comeMI, and W256lescNM.

FIGURE 5

Maximum likelihood tree based on ITS sequences. The following labels apply to samples with identical haplotypes: Trichobilharzia querquedulae TqA= W135blteLA, W137blteLA, W156blteNM, W148.1citeNM, W155.3citeNM, W180citeCA, W203noshAK, W183noshCA, SDS1006noshNE. Trichobilharzia physellae TpB = W146PhysaNM, W263PhysaNM, W171lescPA, W212lescAK, W249cabaNV, W255buheNM. Trichobilharzia franki TfC = Trichobilharzia sp. C, T. franki Ra1, and T. franki RSFO1. All haplotypes of T. regenti downloaded from GenBank were identical; TrD = T. regenti Cz79, T. regenti Cz31, T. regenti Pl27, T. regenti Pl20, T. regenti Pl17, T. regenti Pl14. Trichobilharzia szidati TsE = T. szidati Tsz, T. szidati Ls5, T. szidati ToA. Trichobilharzia brantae TbF = W346GyraulusMB, W331GyraulusCO, W330GyraulusCO. Isolates of T. franki are from R. ovata (ov) and R. auricularia (Ra) snails (one sample is from Lymnaea stagnalis = Ls). The “*” indicates node support of >95% bootstrap for MP and ME and >98 Bayesian PP. The ‘-’ indicates no significant node support.

FIGURE 6

Maximum likelihood tree based on ITS1 sequences of an expanded Clade Q. Trichobilharzia A is boxed to show its variable position in the tree and paraphyly of T. franki. Isolates of T. franki are from R. ovata (ov) and R. auricularia (Ra) snails (one sample is from Lymnaea stagnalis = Ls). See Table III for label descriptions. The “*” indicates node support of >95% bootstrap for MP and ME and >98 Bayesian PP. The ‘-’ indicates no significant node support.

FIGURE 7

Maximum likelihood tree based on ITS2 sequences showing the positions of the unidentified avian schistosome isolates from GenBank. The boxed clade highlights the relationship between the samples from North America and France. Bolded samples indicate those from this study. See Table III for label descriptions. The “*” indicates node support of >95% bootstrap for MP and ME and >98 Bayesian PP. The ‘-’ indicates no significant node support.

FIGURE 8

Summary tree based on 28S depicting comparative features (hosts, distribution, and egg morphology) for North American and European avian schistosomes. Morphological features listed for well-supported nodes. 1– reduced sexual dimorphism, males and females flattened or thread-like, gynaecophoric canal absent or weakly developed or short (not extending to posterior), testes numerous; 2 – Absence of ventral sucker, absent or weakly developed oral sucker, uterus with numerous eggs, eggs ovoid; 3 – Well developed oral and ventral suckers, uterus usually with single egg, seminal vesicle between gynaecophoric canal and ventral sucker; 4 – cecal reunion at or anterior to seminal vesicle, >400 testes, gynaecophoric canal terminates well anterior to first testes; 5 – cecal reunion posterior to gynaecophoric canal, >400 testes, gynaecophoric canal terminates well anterior to first testes; 6 – Position of the cecal reunion overlaps the position of the seminal vesicle, gynaecophoric canal terminates at first testes, cercariae large with eyespots. Physidae, Lymnaeidae, Planorbidae, teal, diving ducks, Anas americana most ducks, swans, geese. Eggs scaled to relative sizes.