The sequences containing the two types of P. marinus SL RNA genes (PmaSLRNAs) were conserved in the first 82-83 nt, with the SL exon of the L type 1-nt longer than that of the S type. Sequence similarity diminished in the downstream intron region. The sequence upstream of SL was more complex: for the L-type PmaSLRNAs, upstream sequences were uniform, whereas those of the S type were diverse, with some resembling the L type ( Figure 1A ). When PmaSLRNAs were aligned with the representatives of known dinoflagellate SL RNAs, PmaSLRNAs showed similarity in the exon (i.e. the 21/22-nt SL region) and moderate similarity in the beginning of the intron region (i.e., immediately downstream; Figure 1B ). As in dinoflagellates, the predicted Smbinding sequence was located in the SL exon of PmaSLRNAs, and the 39 termini of the majority of substrate transcripts mapped within poly-T tracts, reminiscent of the termination element in SL RNAs of kinetoplastid [22] , some dinoflagellates [9] , and of other small RNA genes. The SL RNAs of two Perkinsus spp. and four dinoflagellates were analyzed by gel electrophoresis and hybridization. Ethidium [9] ; the number of identical clones retrieved for each type is indicated by ''@number'' following the species abbreviation and type number). The SL region (boxed) is shown in uppercase letter, intron and the flanking regions are shown in lowercase letters, * indicates the conserved nucleotide (nt). The first 'A' of SL is numbered as nt 1. SL RNA transcripts mapped by 39 RACE analyses are denoted by arrows to indicate the terminal positions, thickness with darkness of the arrows denote relative frequency of clones that ends where it is indicated. Note that the PCR-amplified Amoebophrya sp. genomic sequences contain only one unit of SL RNA gene, the partial SL sequence is of the primer used. Per, P. marinus, Amo, Amoebophrya sp.; Har, Heterocapsa arctica; Kbr, Karenia brevis; Kve, Karlodinium veneficum; Ppi, Pfiesteria piscicida; Pgl, Polarella glacialis; Pmi, Prorocentrum minimum. SL refers to SL RNA sequences obtained from SL-only repeats; SL-5S indicates SL RNA sequences from genes associated with 5S rRNA genes. *: sequences from [8] ; **: sequence from [46] ; #: sequences from [9] , $1-4: GQ178071-GQ178074; N: sequences missing in the original reports. Shaded are conserved positions defined as identical in over six sequences in at least three species. A non-canonical C in the splice donor site of KbrSL-3 is boxed. Gaps introduced in the sequence alignment are shown as '-'. doi:10.1371/journal.pone.0019933.g001 bromide staining revealed that the two Perkinsus species have similar small RNA molecule profiles with commonalities to the dinoflagellate P. minimum (Figure 2A) . Hybridization of an RNA blot of this gel with the 19-nt dinoflagellate SL probe DinoSLa/s (including 14 nt of SL and 5 nt of intron; Table 1 ) showed the dinoflagellate SL RNA pattern with major transcripts of ,56 nt for the four dinoflagellates as reported previously [8] , [9] ; no hybridization was detected for the two Perkinsus species ( Figure 2B ). Probing the blot with P. marinus L-type or S-type SL probes (PmaSL-La/s and PmaSL-Sa/s respectively; Table 1 ), strong bands of .72 nt appeared in both Perkinsus species for both probes, with a minor band of slightly shorter length in the P. marinus sample for probe PmaSL-Sa/s; neither probe hybridized to dinoflagellate SL RNA ( Figure 2C, 2D) , indicating that the .72-nt bands are specific to the genus Perkinsus, and that Perkinsus SL RNAs are longer than those of typical dinoflagellates. Consistent with the similar RNA levels seen on the gel for the two Perkinsus species, probe PmaSL-La/s detected equivalent levels of this SL RNA variant ( Figure 2C ) in the two species. However, the band of P. chesapeaki was weaker than that of P. marinus with probe PmaSL-Sa/s ( Figure 2D ), possibly reflecting reduced expression or impaired hybridization due to a nucleotide alteration(s) in the exon region in P. chesapeaki. The minor band in the P. marinus lane may represent degraded SL RNA products. To further distinguish the two types of PmaSL RNA transcripts and to explore whether P. chesapeaki SL RNAs have similar introns to those of P. marinus, additional probes were designed for the PmaSLRNA L-type and S-type intron sequences (PmaSL-Li and PmaSL-Si; Table 1 ). Both Table 4 . Genomic sequences containing SL RNA genes identified from P. marinus genome data. intron probes revealed bands at .72 nt and some minor bands of ,72 nt in P. marinus ( Figure 2E, 2F ), but no bands in P. chesapeaki, suggesting that P. chesapeaki SL RNAs have different intron sequences than P. marinus. An additional band appeared at ,150 nt with PmaSL-Si for both Perkinsus spp. (Figure 2F ), a likely result of non-specific hybridization to the abundant 5.8S ribosomal RNA (Figure 2A) . To validate the specificity of the probes, 39 RACE cDNA clones of the L-and S-type SL RNA were used to create dot blots that were hybridized separately with each probe. Each yielded a positive signal only when the corresponding probe was used ( Figure 2G, 2H) . A 39 RACE analysis gave an assortment of 39 ends for both PmaSLRNAs. Of the 48 PmaSLRNA cDNA clones mapped, 25 ended at the 2 nd T, 11 clones at the 1 st T, and 4 clones ended at the 3 rd T of the poly-T tracts present in both SL genes, representing 83% of the ends obtained. Thus, most PmaSLRNA transcripts were 80-83 nt in length, corresponding to the major band observed in the RNA blots. The minor end classes of ,72 nt may have contributed to the minor products seen by RNA blotting, possibly representing degraded or misprocessed SL RNA products.