Identification of a divergent environmental DNA sequence clade using the phylogeny of gregarine parasites (Apicomplexa) from crustacean hosts

Abstract

Background: Environmental SSU rDNA surveys have significantly improved our understanding of microeukaryotic diversity. Many of the sequences acquired using this approach are closely related to lineages previously characterized at both morphological and molecular levels, making interpretation of these data relatively straightforward. Some sequences, by contrast, appear to be phylogenetic orphans and are sometimes inferred to represent "novel lineages" of unknown cellular identity. Consequently, interpretation of environmental DNA surveys of cellular diversity rely on an adequately comprehensive database of DNA sequences derived from identified species. Several major taxa of microeukaryotes, however, are still very poorly represented in these databases, and this is especially true for diverse groups of single-celled parasites, such as gregarine apicomplexans.

Methodology/principal findings: This study attempts to address this paucity of dna sequence data by characterizing four different gregarine species, isolated from the intestines of crustaceans, at both morphological and molecular levels: Thiriotia pugettiae sp. n. from the graceful kelp crab (Pugettia gracilis), Cephaloidophora cf. communis from two different species of barnacles (Balanus glandula and B. balanus), Heliospora cf. longissima from two different species of freshwater amphipods (Eulimnogammarus verrucosus and E. vittatus), and Heliospora caprellae comb. n. from a skeleton shrimp (Caprella alaskana). SSU rDNA sequences were acquired from isolates of these gregarine species and added to a global apicomplexan alignment containing all major groups of gregarines characterized so far. Molecular phylogenetic analyses of these data demonstrated that all of the gregarines collected from crustacean hosts formed a very strongly supported clade with 48 previously unidentified environmental DNA sequences.

Conclusions/significance: This expanded molecular phylogenetic context enabled us to establish a major clade of intestinal gregarine parasites and infer the cellular identities of several previously unidentified environmental SSU rDNA sequences, including several sequences that have formerly been discussed broadly in the literature as a suspected "novel" lineage of eukaryotes.

Figure 1. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Cephaloidophora cf. communis.

a. Trophozoite showing the cell organization of the septate gregarine. The cell is divided by a septum (arrow) into the protomerite (PM) with the epimerite (E) at the anterior end and the deutomerite (D) with the spherical nucleus (N). b. An association of two gregarines paired up in caudo-frontal syzygy. The anterior trophozoite is the primite (P), while the posterior trophozoite is the satellite (S). c. SEM showing a trophozoite with epimerite (E), protomerite (PM) and deutomerite (D). Except for the mucron the trophozoite is covered with epicytic folds (arrowheads). There is an indentation (arrowhead) visible at the level of the septum, but the epicytic folds are continuous throughout the trophozoite. d. Higher magnification SEM of the anterior end of a trophozoite with the epimerite (E) free of epicytic folds. There is a visible junction between the protomerite with folds and the epimerite without folds (arrow). Surface pores (double arrowhead) are evenly distributed across the epimerite. Scale bars: Figs. 1a–b, 10 µm; Fig. 1c, 3 µm; Fig. 1d, 1 µm.

Figure 2. Differential interference contrast (DIC) light micrograph and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Heliospora caprellae comb. n.

a. DIC micrograph showing the epimerite (arrowhead), small and rounded protomerite (PM) and the elongated deutomerite (D). The arrow marks the septum between the protomerite and deutomerite. The nucleus (N) is located in the middle of the deutomerite. b. SEM of a single trophozoite with a small protomerite (PM) and a long deutomerite (D) that is wider at the posterior end than at the anterior end. The deutomerite ends in a blunt posterior tip. A slight indentation (arrow) is visible between the protomerite and deutomerite in the area of the septum. Epicytic folds cover the whole trophozoite except for the epimerite (arrowhead) and show an undulating pattern. c. SEM of an association consisting of two trophozoites (or gamonts). The anterior primite (P) has a visible indentation in the area of the septum (arrow) and connects to the anterior end (arrowhead) of the posterior satellite (S). d. Higher magnification SEM of the junction between primite (P) and satellite (S). Some of the epicytic folds (double arrowhead) terminate before they reach the posterior end of the primite. e. High magnification SEM of the epicytic folds. The density of the folds is 5 folds/micron. Scale bars: Fig. 2a, 20 µm; Fig. 2b, 14 µm; Fig. 2c, 11 µm; Fig. 2d, 2 µm; Fig. 2e, 1 µm.

Figure 3. Light micrograph (LM) and scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Heliospora cf. longissima.

a. LM of a trophozoite with an elongated deutomerite (D), a short protomerite (PM), and an epimerite (arrowhead). The septum (arrow) is clearly visible. The spherical nucleus (N) is situated in the middle of the deutomerite. b. SEM of an association of two trophozoites in caudo-frontal syzygy. The junction (arrow) between primite (P) and satellite (S) is visible. The arrowhead marks the epimerite at the anterior end of the primite. c. Higher magnification SEM of the anterior end of the trophozoite with a bulb-like protomerite (PM). The epimerite (arrowhead) shows a prominent collar-like margin (arrowhead). Epicytic folds (double arrowhead) cover the protomerite and deutomerite. d. High magnification SEM of the epicytic folds (double arrowheads). The density of folds is around 3 folds/micron. e. Higher magnification SEM of the area between the primite (P) and satellite (S) of an association of two trophozoites. The junction (arrow) between the two trophozoites is clearly visible. Scale bars: Fig. 3a, 20 µm; Fig. 3b, 10 µm; Figs. 3c–e, 3 µm.

Figure 4. Differential interference contrast (DIC) light micrographs showing the general morphology of the gregarine Thiriotia pugettiae sp. n.

a. DIC micrograph showing an association of two trophozoites. Trophozoite 1 (T1) is longer than trophozoite 2 (T2). The arrowhead marks the slightly broadened anterior tip with the mucron area free of amylopectin. The ellipsoidal nucleus (double arrowheads) is located in the anterior third of the cell in both trophozoites. The attachment site (arrow) of trophozoite 2 is at the level of the nucleus of trophozoite 1. b. Higher magnification view of the anterior end of an association consisting of three trophozoites. The attachment sites (arrows) of trophozoite 2 (T2) and trophozoite 3 (T3) are right behind the nucleus (double arrowhead) of trophozoite 1 (T1). c. Higher magnification view of an association consisting of two smaller trophozoites. The attachment site (arrow) of the much smaller trophozoite 2 (T2) is at the level of the nucleus (double arrowhead) of trophozoite 1 (T1). d. DIC micrograph of two trophozoites. The attachment site of trophozoite 2 (T2) is marked by an arrow. The posterior half of trophozoite 1 (T1) is curled up (arrowhead), a condition that was often recognized when trophozoites were covered in host gut material. e. DIC micrograph of one of the smallest documented trophozoites in an association. The attachment site (arrow) of very small trophozoite 2 (T2) is right behind the nucleus (double arrowhead) of trophozoite 1 (T1). Trophozoite 2 (T2) was only 40 µm long. Scale bars: Fig. 4a, 200 µm; Fig. 4b, 100 µm; Fig. 4c–d, 150 µm; Fig. 4e, 80 µm.

Figure 5. Scanning electron micrographs (SEM) showing the general morphology and surface ultrastructure of the gregarine Thiriotia pugettiae sp. n.

a. SEM of an association of two trophozoites or gamonts in latero-frontal syzygy. The attachment site (arrow) of trophozoite 2 (T2) is located in the anterior half of trophozoite 1 (T1). The arrowhead marks the anterior mucron. b. Higher magnification SEM of the attachment site (arrow) of the two trophozoites. The double arrowhead marks knob-like structures on the trophozoite surface that is inferred to be secreted cell material associated with cell decay. c. High magnification SEM of the attachment site showing a smooth junction (arrow) between trophozoite 1 (T1) and trophozoite 2 (T2). d. SEM of the posterior end of a trophozoite. The trophozoite itself is covered in epicytic folds, but the posterior rounded tip is free of folds. Knob-like structures (double arrowheads) on the surface of the posterior tip is inferred as secreted material associated with cell decay. e. Higher magnification SEM of the anterior end of a trophozoite showing a mucron area (arrowhead) that is broadened and flattened due to a previous attachment to another trophozoite. The cell is also showing the knob-like structures of extruded cell material. f. High magnification SEM of the anterior end of a trophozoite showing the mucron (arrowhead) free of epicytic folds, while the rest of the trophozoite is entirely covered in epicytic folds. Double arrowheads mark the knob-like structures of extruded cell material. Restricted to the anterior end are some larger (super) folds (arrows) in the cell cortex. Scale bars: Fig. 5a, 60 µm; Fig. 5b, 5 µm; Fig. 5c, 1 µm; Figs. 5d–f, 5 µm.

Figure 6. Maximum likelihood (ML) tree of apicomplexans inferred using the GTR + Γ + I model of substitution on an alignment of 82 small subunit (SSU) rDNA sequences and 1,006 unambiguously aligned sites (-ln L  =  17763.96233, α  =  0.514, proportion of invariable sites  =  0.099, eight rate categories).

Numbers at the branches denote ML bootstrap percentage (top) and Bayesian posterior probabilities (bottom); values provided are higher than 65% (i.e., the absence of values reflect statistical support below 65%). Black dots on branches denote Bayesian posterior probabilities and bootstrap percentages of 95% or higher. The six sequences derived from this study are highlighted with black boxes.

Figure 7. Bayesian inference (BI) tree of the studied crustacean gregarines and 48 related environmental sequences inferred using alignment of 1486 unambiguously aligned sites.

Numbers at the branches denote Bayesian posterior probabilities (front) and ML bootstrap percentage (back). Black dots on branches denote Bayesian posterior probabilities and bootstrap percentages of 95% or higher. Black triangles denote groups of highly-similar sequences, numerals inside the triangles denote the number of members of such groups. The environmental sequences shown on Fig. 6 are bolded. Names of the identified gregarine species from crustacean hosts are red, samples from marine environments are blue (light-blue  =  planktonic; cyan  =  benthic), samples from the guts of marine invertebrates are purple (light-purple  =  obtained as common gut contents; dark-purple  =  obtained as identified specific gut parasites), estuarine samples are olive and samples from freshwater environments are green. The four subclades of the crustacean gregarines are color-coded according to their putative family affiliation.