Present Status of the Molecular Ecology of Kathablepharids

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Protist, Vol. 157, 7—11, January 2006 http://www.elsevier.de/protis Published online date 20 January 2006

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In the last few years, environmental sequence parsimony and maximum-likelihood phylogenetic surveys have identified several putative novel analyses including all available kathablepharid lineages within the eukaryotes. This interpretation and kathablepharid-like sequences revealed their is however limited by the absence of taxon sister relationship to cryptophytes (Fig. 1). This sampling for several well known and abundant relationship is congruent with previously published eukaryotes (Cavalier-Smith 2004; Simpson and phylogenies of cultured kathablepharids (Okamo- Roger 2004). It is therefore important to comple- to and Inouye 2005) as well as environmental ment classical morphological studies with mole- studies (Massana et al. 2004; Richards et al. 2005; cular sequencing, as well as with data available Sˇ lapeta et al. 2005). Our phylogenetic reconstruc- from the growing database of environmental tion uncovered three distinct lineages within the phylotype sequences. One of those neglected kathablepharid sequences (Fig. 1). The first clade but abundant protist groups are the kathablephar- grouped the marine species L. marina with five ids (Kathablepharida — ICZN, Katablepharido- marine phylotypes, including the partial Mediter- phyta — ICBN); predatory heterotrophic ranean phylotype BL000921.34, which is 499% flagellates that have escaped molecular sequen- identical to L. marina. The other four phylotypes cing until very recently. Kathablepharid flagellates were obtained from studies of Western North are a significant component of freshwater and Atlantic and Spanish Mediterranean coastal sam- coastal food webs (Arndt et al. 2000), but their ples (Countway et al. 2005; Massana et al. 2004), relationship to other eukaryotic groups remained and were 97% identical to L. marina. The uncertain, although most authors suggested their second lineage comprised a single representative, affinity with the cryptophytes (for review see the marine species K. japonica. It formed a well- Okamoto and Inouye 2005). supported cluster with the L. marina clade (Fig. 1), In a previous publication we reported the although visual inspection of the alignment re- environmental SSU rDNA survey of a freshwater vealed clear differences that justify the designa- pond, where we identified an unknown phylotype tion of two independent lineages. The third clade distantly related to the cryptophytes (Sˇ lapeta et al. forms a sister group to the previous two clades. It 2005). We hypothesised that this phylotype could comprised only phylotypes from freshwater en- belong to the kathablepharids, a group not vironments, with no related sequence from mor- represented in sequence databases at that time. phologically described taxa. These phylotypes are The proposed identity remained unsettled until a all 99% identical to each other and 92 to 94% recent study by Okamoto and Inouye (2005) identical to the two other kathablepharid clades. provided the first SSU rDNA sequences from These results show a widespread presence of two well-characterised marine kathablepharid kathablepharids in marine and freshwater sam- species, Kathablepharis japonica and Leucocryp- ples, which is in agreement with morphology- tos marina. Phylogenetic analyses based on the based studies that identify kathablepharids as SSU rDNAs of these two species as well as b- predominant among the heterotrophic nanoflagel- tubulin sequences indicated their sister relation- lates (Arndt et al. 2000). However, we observe a ship to cryptophytes (Okamoto and Inouye 2005). distinct grouping of marine and freshwater phylo- Using the published SSU rDNA sequences of K. types, suggesting the existence of specific clades japonica and L. marina (Okamoto and Inouye adapted to these environments. In particular, 2005) as queries for BLAST searches, we identi- the recurrent identification of kathablepharid fied 11 phylotypes very similar to these katha- sequences in the three freshwater SSU rDNA blepharid sequences (see Table 1). Maximum surveys suggests that freshwater kathablepharids

8 J. Sˇ lapeta et al. lapeta et al. (2005) ˇ S Lefranc et al. (2005) Lefranc et al. (2005) Richards et al. (2005) Richards et al. (2005) Richards et al. (2005) Okamoto and Inouye (2005) Okamoto and Inouye (2005) Massana et al. (2004) Massana et al. (2004) Countway et al. (2005) Countway et al. (2005) ). AY919743 , gi: 60476994), for details about the origin see the text. AY919790 m filtrate. m 5 4 FreshwaterFreshwater Lake LaFreshwater Godivelle lac Lake d’en Pavin, haut, Massif Massif Central, Lake Central, France George, France Adirondack Park, USA Freshwater Chevreuse Pond, Ile-de-France, France FreshwaterFreshwater Lake George, Adirondack Park, Lake USA George, Adirondack Park, USA Marine Tokyo Bay, Japan MarineMarineMarine Tokyo Bay, Japan MarineMarine Barnegat Bay, NewMarine Jersey, Blanas USA Bay, Mediterranean coast, Blanas Spain Bay, Mediterranean coast, Atlantic Spain Ocean, western north Atlantic Atlantic Ocean, western north Atlantic Lim and Vandergrift (unpubl.) , gi: 60476971) is a chimerical sequence composed of kathablepharid + ciliate; 1296—1696 nt are most , gi: 60476910) is a chimerical sequence composed of chrysophyte + kathablepharid; 1—559 nt are most AY642732 AY642702 AY919689 AY821954 AY919706 AY919767 AB231617 AB193602 AB194980 AY884991 AY426851 AY426929 AY938299 AY938049 AY919767 AY919706 Ã ). These clones were recovered from water filtrates from three different libraries; 6 clones from 2 libraries constructed from ÃÃ m filtrates and 2 clones from y m Summary of available sequences related to kathablepharids Phylotype LG24-02 ( Phylotype LG08-04 ( Ã ÃÃ lapeta et al. 2005 ˇ The phylotype CH1_5A_4 represents a fully sequenced clone randomly chosen out of 8 closely related and partially sequences clones S closely related to the sequence of a phylotype LG31-02 ( ( 5 to 0.22 closely related to the sequence of a chrysophyte (stramenopiles, Heterokonta) phylotype LG18-09 ( PG5.31 P34.10 LG04-01 LG08-04 (560—1708) LG24-02 (1—1295) Table 1. Name/PhylotypeKathablepharis japonica Acc. nos. Environment Localityy Reference Leucocryptos marina BB01-16 BL000921.34 BL010625.24 ENI47296.00145 ENI42482.00159 CH1_5A_4 ARTICLE IN PRESS

Molecular Ecology of Kathablepharids 9

CH1_5A_4 AY821954 freshwater

PG5.31 AY642732 clade Kathab 99/100 LG24-02 * AY919767 (1-1295 nt) LG04-01 AY919689 LG08-04 * AY919706 (560-1708 nt) 100/100 P34.10 AY642702

lepharid Kathablepharis japonica AB231617 marine BL000921.34 AY426851 100/100 89/8 clades BB01-16 AY884991 9 Leucocryptos marina AB193602+AB194980 85/92 BL010625.24 AY426929

a ENI42482.00159 AY938049 91/93 ENI47296.00145 AY938299

93/86 Hemiselmis brunnescens AJ007282 Cryptophyta 64/- Komma caudata U53122 90/83 Telelaulax acuta AF508275 Rhodomonas mariana X81373 50/- Cryptomonas platyuris AF508271 Proteomonas sulcata AJ007285 Goniomonas truncata U03072 98/97 Goniomonas pacifica AF508277 BL000921.41 AY426853 100/100 Cyanophora paradoxa X68483 Glaucophyta Glaucocystis nostochinearum X70803 0.05

Figure 1. Phylogenetic reconstruction of SSU rDNA kathablepharid and kathablepharid-like sequences retrieved from environmental studies. The two published kathablepharid SSU rDNA sequences, Kathablepharis japonica and Leucocryptos marina (Okamoto and Inouye 2005), served as queries for reciprocal BLAST searches, and multiple sequence alignments were constructed using the Clustal algorithm. Chimerical sequences were identiﬁed manually. Multiple sequence alignments were inspected by eye using true kathablepharid sequences as proxies to the identity of selected sequences. Varying sections of the sequences were (1) eliminated from preliminary phylogenetic reconstructions and (2) used in BLAST searches as queries. To decipher the identity of the non-kathablepharid sequence regions, we retrieved the sequences corresponding to the highest scoring BLAST hits and constructed multiple sequence alignments including the full chimerical sequence to validate the break point within the sequence. Phylogenetic trees were reconstructed using maximum parsimony with MEGA3 (Kumar et al. 2004) and maximum likelihood with PhyML 2.4.4. (Guindon and Gascuel 2003). Distance methods were not used due to their inferior capacity to reconstruct correct relationships of taxa when sequences are partial. After elimination of the ambiguous parts of the original multiple sequence alignment (2042 residues), the ﬁnal alignment contained 1601 residues. The phylogenetic tree shown was reconstructed using the maximum likelihood method (GTR+G+I model with parameters estimated) and rooted with two glaucophyte sequences. Bootstrap support (100 resamplings) for maximum likelihood/maximum parsimony is indicated above the branches. Accession numbers are indicated to the right. Alignments are available from the authors upon request. Phylotypes with ‘*’ indicate original chimerical sequences, non-kathablepharid regions were excluded from analysis, the nucleotides retained are listed in parentheses (for details see Table 1).

are rather frequent. To have a precise taxonomic of phylotype sequences with better taxon identiﬁcation of these phylotypes, it is urgent to sampling to rule out contaminating chimerical sequence the SSU rDNAs of freshwater Katha- sequences. The recent availability of true blepharis spp., including the type species as well kathablepharid sequences opened the opportu- as the third kathablepharid genus known, Platy- nity to update the phylotype diversity and critically chilomonas (Patterson et al. 2000). reanalyse the kathablepharid-like sequences. An While environmental sequence surveys have additional problem frequently encountered with generated a number of interesting phylotype environmental phylotypes is the sequencing of sequences, several of those indicated as repre- only part of the SSU rDNA gene, with the region sentative of ‘novel’ major clades need further sequenced differing very often from study to scrutiny (Berney et al. 2004; Cavalier-Smith 2004). study. For example, the ﬁve marine phylotypes In this sense, Berney et al. (2004) have paid related to L. marina are only 600 to 700 bp long. special attention to the need for a careful analysis Due to the different SSU rDNA primers used, the ARTICLE IN PRESS

10 J. Sˇ lapeta et al. relationship of BB01-16 to the other phylotypes BL000921.41 (from Mediterranean coastal water) would have never been recovered without the branched, although with weak bootstrap support, existence of the complete L. marina sequence. at the base of the cryptophytes (Fig. 1), which The previous absence of bona fide kathable- suggested that an intermediate clade between the pharid sequences rendered the identification of cryptophytes and the kathablepharids may exist. chimerical phylotypes difficult. We have now The identification of additional sequences belong- reanalysed the 11 kathablepharid-related phylo- ing to this clade will be necessary to ascertain this types and identified two chimeras. Both come hypothesis. from a recent survey of freshwater picoeukaryotes Molecular ecology and classical techniques are in the oligotrophic southern basin of Lake George complementary approaches to map the biodiver- (Richards et al. 2005). The authors retrieved three sity of protists. However, thorough revision of ‘distinct’ phylotypes clustering into a clade with a environmental sequences is critical to make any close affinity either to cryptophytes or glauco- safe conclusions on protist diversity and phylo- phytes (clade ‘LG-E’ in their work). For these three geny, as errors introduced during the production phylotypes, the best BLAST matches corre- of the sequences (PCR amplification, sequencing, sponded to the known kathablepharids K. japoni- sequence assemblage) may mask the true phylo- ca and L. marinus. Nevertheless, a closer geny or incorrectly inflate the number of different inspection of their sequences and comparison protist groups. It is therefore imperative to check with those from the database revealed that two of the quality of individual sequences before inter- these three phylotypes are of chimerical origin. preting phylogenetic trees. This is particularly The phylotype LG08-04 turned out to be a crucial for those environmental phylotypes for chimerical sequence composed of a chryso- which no reference organisms have been se- phyte-like 50 region and a true kathablepharid 30 quenced. region. The chrysophyte-like fragment was almost identical to the sequence of the phylotype LG18- 09 from the same study (for details see figure 2 in Richards et al. 2005). The origin of LG24-02 was Acknowledgement more complex. The true kathablepharid-like fragment represented only the 50 region, being This work was supported by an ATIP grant of the identical or almost identical to the phylotypes French Centre National de la Recherche Scienti- within the freshwater clade defined in our phylo- fique, section ‘Dynamique de la biodiversite´ ’. genetic analysis (Fig. 1). The 30 region of LG24-02 was most closely related to the Lake George phylotype LG31-02, originally reported as an unknown heterokont related to chrysophytes. References However, we found that this phylotype LG31-02 (sequence accession number AY919790) was also Arndt H, Dietrich D, Auer B, Cleven E-J, Grafen- a chimerical sequence composed of a 50 chryso- han T, Weitere M, Mylnikov AP (2000) Functional phyte-like region (499% identity with Dinobryon Diversity of Heterotrophic Flagellates in Aquatic Ecosystems. In Leadbeater BSC, Green JC (eds) sertularia, AF123289) and a 30 ciliate-like part. The Flagellates: Unity, Diversity and Evolution. Taylor Interestingly, only the ciliate-like region of LG31-02 & Francis, London, pp 240—268 is identical to the 30 region of LG24-02 (see Table 1). BLAST searches with only this 30 region as a Berney C, Fahrni J, Pawlowski J (2004) How many query did not reveal a very close relationship to novel eukaryotic ‘kingdoms’? Pitfalls and limitations any known ciliate sequence, but only 95 to 96% of environmental DNA surveys. BMC Biology 2:13 identity to several spirotrichean and oligohyme- Cavalier-Smith T (2004) Only six kingdoms of life. nophorean ciliates (e.g. Strobilidium caudatum Proc R Soc Ser B 271: 1251—1262 AY143573 and Tintinnopsis tubulosoides AF399111). When we excluded the non-kathable- Countway PD, Gast RJ, Savai P, Caron DA (2005) pharid regions of the two phylotypes LG24-02 and Protistan diversity estimates based on 18S rDNA LG08-04, they proved to be 499% identical. It is from seawater incubations in the Western North very probable that the contaminating parts of Atlantic. J Eukaryot Microbiol 52: 95—106 these sequences masked their sister relationship Guindon S, Gascuel O (2003) A simple, fast, and to the cryptophytes in the original study (Richards accurate algorithm to estimate large phylogenies by et al. 2005). Interestingly, the phylotype maximum likelihood. Syst Biol 52: 696—704 ARTICLE IN PRESS

Molecular Ecology of Kathablepharids 11

Kumar S, Tamura K, Nei M (2004) MEGA3: Richards TA, Vepritskiy AA, Gouliamova DE, Integrated software for molecular evolutionary ge- Nierzwicki-Bauer SA (2005) The molecular diversity netics analysis and sequence alignment. Brief Bioin- of freshwater picoeukaryotes from an oligotrophic form 5: 150—163 lake reveals diverse, distinctive and globally dis- persed lineages. Environ Microbiol 7: 1413—1425 Lefranc M, Thenot A, Lepere C, Debroas D (2005) Genetic diversity of small eukaryotes in lakes Simpson AG, Roger AJ (2004) The real ‘kingdoms’ differing by their trophic status. Appl Environ Micro- of eukaryotes. Curr Biol 14: R693—R696 biol 71: 5935—5942 Sˇ lapeta J, Moreira D, Lo´ pez-GarcıáP(2005) The Massana R, Balague V, Guillou L, Pedro´ s-Alio´ C extent of protist diversity: insights from molecular (2004) Picoeukaryotic diversity in an oligotrophic ecology of freshwater eukaryotes. Proc R Soc Ser B coastal site studied by molecular and culturing 272: 2073—2081 approaches. FEMS Microbiol Ecol 50: 231—243 Okamoto N, Inouye I (2005) The katablepharids are ˇ 1 a distant sister group of the Cryptophyta: a proposal Jan Slapeta , Purificacio´ nLo´ pez-Garcıá, and for Katablepharidophyta Divisio Nova/Kathablephar- David Moreira ida Phylum Novum based on SSU rDNA and Beta- Unite´ d’Ecologie, Syste´ matique et Evolution, UMR tubulin phylogeny. Protist 156: 163—179 CNRS 8079, Universite´ Paris-Sud, Baˆ t. 360, 91405 Orsay Cedex, France Patterson DJ, Vørs N, Simpson AGB, O’Kelly C (2000) Residual Free-Living and Predatory Hetero- 1Present address and address of correspondence: Institute trophic Flagellates. In Lee JJ, Leedale GF, Bradbury for the Biotechnology of Infectious Diseases, University of P (eds) An Illustrated Guide to the Protozoa. 2nd Technology, Sydney, Westbourne Street, Gore Hill, NSW 2065, edn. Society of Protozoologists, Lawrence Kansas, Australia fax 61 2 9514 4201 pp 1302—1328 e-mail [email protected] (J. Sˇ lapeta)