Refining cryptophyte identification with DNA ... - Oxford Journals

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Oct 10, 2007 - ALFRED WEGENER INSTITUTE FOR POLAR AND MARINE ..... Eppley, R. W., Holmes, R. W. and Strickland, J. D. H. (1967) Sinking rates of ...
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Refining cryptophyte identification with DNA-microarrays KATJA METFIES AND LINDA K. MEDLIN* ALFRED WEGENER INSTITUTE FOR POLAR AND MARINE RESEARCH, AM HANDELSHAFEN

12, D-27570 BREMERHAVEN,

GERMANY

*CORRESPONDING AUTHOR: [email protected] Received May 8, 2007; accepted in principle July 19, 2007; accepted for publication October 9, 2007; published online October 10, 2007 Communicating editor: K.J. Flynn

The division Cryptophyta, Class Cryptophyceae, contains ecologically important species that are found in all kinds of aquatic habitats. The identification of the Cryptophyta is challenged by a need to examine species in the SEM or TEM to visualize features needed to identify its species. Thus, for routine monitoring programmes, this group is not identified beyond the level of class and that identification is only done if the samples are routinely examined with a fluorescent microscope and the cryptophytes are counted based on the natural orange fluorescent of their phycobilin pigments. We present a set of molecular probes that target the 18S rDNA of the class Cryptophyceae and its major clades. This includes two probes at the level of class and 12 probes to distinguish the different clades, which should be amended to the level of family or genus. Within the Cryptophyceae, seven clades have been described previously, which do not correspond to the present day classification of the group. The probes can be used for a multiplexed analysis on a PHYLOCHIP, which allows a rapid characterization of Cryptophyceae in field samples. The probes have been tested for specificity and their applicability on a DNA-microarray proven.

I N T RO D U C T I O N The division Cryptophyta consists of biflagellate unicellular microalgae with a remarkable diversity with a unique morphology and pigmentation, although there are colourless members, which are primitive in their rDNA phylogeny (Hoef-Emden et al., 2002). The colourless and pigmented forms represent the two classes of the division. The division contains 200 species assigned to more than 20 genera, which have been newly relegated into orders and families based on a preliminary rDNA tree presented by Marin et al. (Marin et al., 1998). The cells are ubiquitously found in marine, brackish and freshwater habitats (Clay et al., 1999). In the light of problems of diversity changes coupled to environmental changes, proper identification to species level is becoming increasingly important to assess changes in biodiversity that go along with the environmental changes. The total concept of biodiversity and the challenge of quantifying that diversity rests heavily on being able to identify clearly species in the community. However, the identification of Cryptophyta is a

challenging task because certain preservatives, such as glutaraldehyde, rupture the cells and species must be examined in the SEM or TEM with cryofixation to visualize features identifying the species (Clay et al., 1999). A new phylogenetic reconstruction of the Cryptophyta, with more taxa, has been inferred from ribosomal genes (Hoef-Emden et al., 2002, Hoef-Emden and Melkonian, 2003), which does not correspond directly to that based on ultrastructural features (Clay et al., 1999), which relies on electron microscopy and the first rDNA tree presented by Marin et al. (Marin et al., 1998). However, the identification of Cryptophyta by electron microscopy or sequencing is time-consuming, labour intensive and costly. To add to the problems, cryptophytes have dimorphic life cycles and there are few studies that have linked the haploid cell stage to the diploid one, both of which are likely to have been identified as separate species/genera (Hill and Wertherbee, 1986) because they had different ultrastructures. These drawbacks could be overcome by the application of DNA-microarray technology, which

doi:10.1093/plankt/fbm080, available online at www.plankt.oxfordjournals.org # The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]

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allows for multiple parallel identification of microorganisms in a single experiment (Metfies and Medlin, 2004) and the simultaneous identification of both stages of the life cycle independent of morphology. Here, we present a set of probes that has been developed for the identification of Cryptophyceae at the class and clade or family level, which greatly enhances the identification of the group. The probe set has been tested for the specificity and for their applicability on a DNA-microarray. As more and more probes are designed and tested for microarrays, the cost-effectiveness of this method becomes more attractive and reasonable.

M E T H O D S , R E S U LT S A N D DISCUSSION The phylogenetic analysis of the nuclear 18S rDNA of the Cryptophyta identified eight clades within the division (Hoef-Emden, 2002). Here, we present a set of molecular probes that allows the identification of cryptophytes according to this classification. The probes were designed using the ARB software package (http://

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www.arb-home.de). The probes have a length of 18, or 25 bases with at least two mismatches against all nontarget organisms. The ARB database contained over 3000 published and unpublished algal sequences. All publicly available 18S rDNA sequences assigned to the division Cryptophyta were downloaded from Genbank (http://www.ncbi.nlm.nih.gov). It was possible to design two probes that targeted all pigmented species that belong to the division Cryptophyta, Class Cryptophyceae (the pigmented species) and 14 probes could be designed that target the different clades within this class recovered by Hoef-Emden et al. (Hoef-Emden et al., 2002). The different clades that are targeted by the probe set are shown in a schematic diagram (Fig. 1). For clade 6, it was only possible to design a probe that also targets clade 4. The probe sequences and their targets are listed in Table I. Originally, we aimed to develop a probe-set that identified the Cryptophyceae down to the genus-level to obtain tools for a highresolution analysis of cryptophyte abundance. However, we found that it was not possible to design highly specific probes for the different genera within the Cryptophyceae. The 18S rDNA of species assigned to

Fig. 1. Schematic drawing to illustrate the targets of the probes described in this publication. This scheme was based on the phylogenetic tree from Hoef-Emden et al. (Hoef-Emden et al., 2003).

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Table I: Probe sequences and targets of the probe set for the identification of the division Cryptophyta on a DNA-microarray. Probe

Sequence

Target

Source

Euk1209 Crypto A Crypto B Crypt 013 Crypt 01 –25 Crypt 02 Crypt 02-25 Crypt 03 Crypt 03 –25 Crypt 04 –25 Crypt 46

GGGCATCACAGACCTG CACTAAGACATGCATGGC ACGGCCCCAACTGTCCCT TCATTACCCCAGTCCCAT CATTACCCCAGTCCCATAACCAACG CATTACCCCAGTCCCAT GTCCCACTACCCTACAGT GCGTCCCACTACCCTACAGT GTCCCACTACCCTACAGTTAAGT TTCCCGCGCACCACGGTT GTGTTCCCGCGCACCACGGTT TTCCCGCGCACCACGGTTAAAT CACCTCCACCATAAAGGCATGAGGT CAAGGTCGGCTTTGCCTC

Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) Metfies et al. (2007) This work Metfies et al. (2007)

Crypt Crypt Crypt Crypt PC NC

GTCCCAACGCCCCTCAGT TGCGTCCCAACGCCCCACAGTGAAG ACAAGGTCGGCTTGAATC CACCAAAACAAGGTCGGCTTGAATC ATGGCCGATGAGGAACGT TCCCCCGGGTATGGCCGC

All Eukaryotes All Cryptophyceae All Cryptophyceae Cryptophytes, clade 1 Cryptomonas Cryptophytes, clade 1 Cryptophytes, clade 2, Rhinomonas, Rhodomonas Cryptophytes, clade 2 Cryptophytes, clade 3, Hanusia, Guillardia Cryptophytes, clade 3 Cryptophytes, clade 4, Plagioselmis, Teleaulax, Geminigera Cryptophytes, clades 4 and 6, Komma, Chroomonas, Hemiselmis, Plagiomonas Cryptophytes, clade 5, Proteomonas Cryptophytes, Clade 5 Cryptophytes, clade 7, Falcomonas Cryptophytes, clade 7 TATA-Box-Binding Protein S. cerevisiae Negative control

053 053 – 25 07 07 –25

Metfies et al. (2007) Metfies et al. (2007) This work This work Metfies et al. (2004) Metfies et al. (2004)

Bases in bold indicate a common motif in the probes.

the genera Campylomonas, Cryptomonas and Chilomonas grouped together in one clade and one probe could be developed that specifically targets this clade, but it was not possible to design a specific probes for the different genera in this clade. In the course of the probe development, a revision of the genus Cryptomonas was published, that could explain this observation. A heteromorphic life cycle for the cryptomonads was first published by Hill and Wetherbee (Hill and Wetherbee, 1986), but such a life cycle has been documented in very few of its species. Using molecular data, the genus Cryptomonas and Campylomonas were linked together, whereas Campylomonas and Chilomonas were reduced to synonyms of the genus Cryptomonas (Hoef-Emden, 2003). Thus, our probe for clade 1 is actually a genus level probe for Cryptomonas because this is now the only genus in this clade. The impossibility of developing genus level probes for the other clades suggest that further revision of the division Cryptophyta could identify pairs of species in the life cycle of the Cryptophyta. The specificity and applicability of the probes in the microarray format were tested by hybridizations of PCR-fragments amplified from the 18S rDNA isolated from unialgal laboratory cultures of Chilomonas sp. CCAP 977/1, Campylomonas reflexa CCMP1177, Rhinomonas reticulata PLY 358, Rhodomonas sp. CCMP 768, Hanusia phi CCMP 325, Guillardia theta CCMP 327, Plagioselmis prolonga CCMP 644, Proteomonas sulcata CCMP 327, Hemiselmis virescens, Chroomonas salina PLY 544. The laboratory cultures were grown at 158C or 208C with a photon flux density of 100 mmol m22 s21 and a

light/dark photocycle of 12:12 h in enriched seawater media K (Keller et al., 1987), IMR (Eppley et al., 1967) or Drebes (Drebes, 1966). After a filtration of the cultures, genomic DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Germany) and used as a template for subsequent PCR-amplification. A fragment of 18S rDNA from the selected species was amplified using the eukaryotic primers 82F-biotin (50 -GTGAAACTGCGA ATGGCTCAT- 30 ) and 1055R (50 -ACGGCCATGC ACCACCACCCAT- 30 ) (Elwood et al., 1985). The primer 82F was labelled with a biotin moiety at the 50 -end of the molecule. The PHYLOCHIPS to test the specificity of the Cryptophytes were purchased either from PicoRapid (Bremen, Germany) or spotted with the GMS 417 Arrayer (Genetic Microsystems, Inc., Woburn, USA). Oligonucleotide probes for microarray printing were obtained from Thermo Hybaid, Interactiva Division (Ulm, Germany) with a C6/MMT Aminolink at the 50 -end of the molecule. The probes were spotted at a concentration of 10 mmol in spotting buffer. Epoxycoated slides for the in house spotting of probes were purchased from Nexterion (Germany). The probes were spotted at a concentration of 10 mM in QMT spotting solution Quantifoil Micro Tools GmbH (Jena, Germany). A saturated solution of BSA-Cy5 was used as a control for the spotting process. Each probe was spotted with six replicates. The spot-size was 150 mm. For efficient cross-linking of the oligonulceotides to the slide-surface, printed DNA-microchips were incubated for 30 min at 608C. After the printing procedure, the DNA microchips were stored at –208C.

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The microarray hybridization was carried out as described by Metfies and Medlin. (Metfies and Medlin, 2004). If possible, the specificity of the clade level probes was assessed by a hybridization to the 18S rDNA of species belonging to two different genera from each clade, using probes of two different lengths. For clade 5, it was only possible to test the specificity with the genus Proteomonas, because this is the only genus within the clade. The probes for clade 7 could only be tested for non-specific binding, because it was not possible to get a culture of Falcomonas daucoides, the only representative of clade 7. The results of the hybridizations are presented in Fig. 2. Two probes, Crypto A and Crypto B, were designed to target all pigmented species within the division Cryptophyta, Class Cryptophyceae. In specificity tests with species from 10 different cryptophyte genera, Crypto B appeared to be specific, but Crypto A gave only false negative signals with a signal intensity that was not significantly above background. The probes of two different lengths contained a common motif and the longer probe was

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extended by three bases at the 50 -end and four bases at the 30 -end of the shorter probe sequence. In the majority of cases, the 25-mers resulted in higher signal intensities than the corresponding 18-mer probes (Metfies et al., 2007). The majority of the probe set for the different clades displayed high specificity. This involves the probes Crypt 01 and Crypt 01– 25 targeting clade 1, Crypt 02 and Crypt 02– 25 targeting clade 2, Crypt 03 and Crypt 03– 25, Crypt 46 targeting the clades 4 and 6, and Crypt 053 and Crypt 053– 25 targeting clade 5. Other probes that we designed for the clades were unspecific when hybridized with the 18S rDNA of non-target species. These involved Crypt 04– 25, which cross-hybridized with all 10 tested target species, Crypt 05, which cross-hybridized to Rhodomonas sp. CCMP 768, and Crypt 07 and Crypt 07– 25, which cross-hybridized to P. prolonga CCMP 644. Reasons for these cross-hybridizations do not lie in the primary structure of the probes but in other factors that are not explainable from our experiments and from in silico tests.

Fig. 2. Specificity tests of the probes in with PCR amplified 18S rDNA fragments in the microarray format. (A) Hybridization of Protoeromonas sulcata. The results show a typical hybridization of a PCR amplified 18S rDNA fragment to a microarray containing the indicated hierarchical probes. The different pictures in A reflect three different blocks on the same DNA-chip, which contained four blocks. The fourth block is not displayed here, because it contained probes not relevant for this publication. One block consisted of the probes indicated at the left side of each picture. The probes were spotted in six replicates in a row. (B) Schematic drawing of the complete specificity test of the probes described here.

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Our study provides a set of probes for a hierarchical identification of the Division Cryptophyta, Class Cryptophyceae, which is important for enhancing the identification of this group of ecologically important phytoplankton. With this probe set, it is now possible to identify crytpophytes to family level and where there is only one genus in the family, the resolution is at the generic level. The set of probes can be used in the microarray format, which allows high throughput analysis of phytoplankton samples. A field sample taken at the island of Helgoland in the German Bight was analysed with a microarray containing the new set of probes to prove applicability of the probes in the field (Metfies et al., 2007) and more studies are underway with these probes and time series data from two locations, Helgoland in the German Bight and Arcachon in the Bay of Biscay. The design of hierarchically organized probe sets for the use in different hybridization formats and the implementation of the probes to a comprehensive PHYLOCHIP for the monitoring of phytoplankton is an on-going approach in our laboratory. The implementation of the present probe set to the PHYLOCHIP would aid significantly to improve the resolution of monitoring phytoplankton occurrence, especially where taxonomic expertise is limited. The probe set gives higher resolution to the analysis of field samples considering the occurrence of Cryptophyceae. At the Arcachon long-term monitoring site, Cryptophyceae are only manually counted in general at the class level based on their fluorescence properties, because the identification at a lower taxonomic level would be too time-consuming in the context of longterm monitoring.

AC K N OW L E D G E M E N T Helga Mehl provided technical assistance.

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FUNDING EU Project MICROPAD QLK3-CT-2002-01939; Stiftung Alfred-Wegener-Institut fu¨r Polar– und Meeresforschung in der Helmholtz-Gesellschaft, Bremerhaven, Germany.

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