BMC Genomics BioMed Central

Research article Open Access The (CYP) superfamily in pulex William S Baldwin*1, Peter B Marko2 and David R Nelson3

Address: 1Clemson University, Biological Sciences, Institute of Environmental Toxicology, 509 Westinghouse Road, PO Box 709, Pendleton, SC, USA, 2Clemson University, Biological Sciences, 132 Long Hall, Clemson, SC, USA and 3University of Tennessee-Memphis, Department of Molecular Sciences, G01 Molecular Science Building, Memphis, TN, USA Email: William S Baldwin* - [email protected]; Peter B Marko - [email protected]; David R Nelson - [email protected] * Corresponding author

Published: 21 April 2009 Received: 13 February 2008 Accepted: 21 April 2009 BMC Genomics 2009, 10:169 doi:10.1186/1471-2164-10-169 This article is available from: http://www.biomedcentral.com/1471-2164/10/169 © 2009 Baldwin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: Cytochrome P450s (CYPs) in fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results: Data mining and manual curation of the Daphnia pulex identified 75 functional CYP , and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome . Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone (Nematostella vectensis) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex, whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans. Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex. Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (). Conclusion: Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan.

Background In addition, the CYP26 family contributes to maintenance The cytochrome P450s (CYPs) have widespread and of sharp retinoic acid boundaries between rhombomeres diverse functions in animals. CYPs in families 1–4 are crit- in the developing mammalian embryo and in the eye [9- ical and often inducible components of the phase I detoxi- 11]. In insects CYPs are essential to the function of sensory fication systems of vertebrates, invertebrates, and plants organs such as antennae, where they may be involved in [1-4]. They are also important in lipid metabolism, odorant clearance [12]. including fatty acids, retinoids, eicosanoids, , vita- min D and bile acids, and some are regulated in a sexually CYPs are important in the metabolism of and tolerance to dimorphic fashion through endocrine mechanisms [5-8]. anthropogenic chemicals [13] and plant allelochemicals

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[14,15]. Inducibility, constitutive overexpression, and tation to environmental challenges, and as biomarkers of genomic studies investigating quantitative trait loci (QTL) chemical or endocrine stimulation. mapping have demonstrated tolerance or resistance to environmental chemicals due to CYPs [13,16,17]. Fur- Results and discussion thermore, it has been hypothesized that pesticide sensitiv- Manual Annotation ity in honeybee is associated with reduced CYP numbers Manual annotation and curation of the CYPs in Daphnia [18]. In addition, hormone structure is dependent on pulex v1.1 draft genome sequence assembly (September, CYPs [19], and therefore small differences in the hor- 2006) produced 73 full length P450s and three pseudo- mones utilized (i.e. juvenile hormones, methyl far- genes. In addition, there are two ESTs in the CYP4 clan nesoate) may be dependent on the CYPs available or the that were not found in the D. pulex genome of the chosen timing of expression. parthenogenic individual, but partial sequences (Cyp4C32, Cyp4AN1) were observed previously in spe- Daphnia pulex, commonly known as the waterflea, is the cific D. pulex ecotypes [15]. Overall, this number is similar first to have its genome sequenced. D. pulex has to other CYP genomes (Additional file 1), but slightly a genome of approximately 200 Mb with 31,000 genes lower than the typical invertebrate or insect with the http://wfleabase.org. Daphnid are globally distrib- exception of honeybee [22]. uted zooplankton, in the order , suborder , and there are several daphnid species, includ- The Daphnia pulex full length CYPs are divided into four ing , , Daphnia pulex, and distinct clans; mitochondrial, CYP2, CYP3, and CYP4, the . Daphnids are commonly studied zoo- same clans that are found in insects [22]. The four clans plankton because of their importance to aquatic ecosys- can be further subdivided into 13 families, 19 sub- tems, ability to contend with environmental challenges, families, and 75 putatively functional isoforms, and 3 amenability to culture, short life-cycle, and parthenogenic pseudogenes (Fig. 1). The phylogenetic tree in combina- reproduction. Furthermore, daphnids are commonly used tion with the previously described rules for naming CYPs in several toxicity tests for multiple applications. Studies (40% identity for family designation, 55% identity for on the Daphnia pulex genome and specifically the CYPs subfamily designation) were used to name the different may provide important insight on how genome and gene expression alterations promote individual and popula- tion-level fitness following environmental change.

It has become clear in the genomic era that previous esti- mates for the number of CYPs in daphnids such as D. magna [20] was a gross underestimate, and that most metazoans have a large number of CYP genes. For exam- ple, humans have 57, mice have 102, Stronglyocentrotus purpuratus (sea urchin) have 120, Anopheles gambiae (mos- quito) have 106, and melanogaster have 83 functional CYPs (Additional file 1). In continuation of this P450 research, we are working with the Daphnia Genomics Consortium to assemble, annotate, and com- pare the CYPs in the Daphnia pulex genome with several other species. We have also assigned names to all of the CYPs and CYP pseudogenes based on previously pub- lished rules using phylogenetic trees and amino acid sequence identity to determine clan, family and subfamily membership [21]. Clans represent the deepest diverging gene clades in the CYP nomenclature. There are ten P450 clans among animals, but only four are present in the pro- tostomes: CYP2, CYP3, CYP4 and mitochondrial. The DaphniaCYP2,Figure CYP3, 1pulex andCYPs CYP4 are divided into fourclans; mitochondrial, general web repository for P450 nomenclature and Daphnia pulex CYPs are divided into fourclans; mito- sequence data is http://drnelson.utmem.edu/ chondrial, CYP2, CYP3, and CYP4. The four clans can cytochromeP450.html. Overall, this work provides a con- befurther subdivided into 13 families, 19 subfamilies, and 78 tinuation of earlier projects to comprehensively annotate isoforms, including pseudogenes. The numbers represented CYPs and determine the putative role of Daphnia pulex above include thepseudogenes, of which there is one pseudo- CYPs in sensory adaptation, sensitivity to toxicants, adap- gene in each of the CYP2, 3, and 4 clans.

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CYPs in D. pulex [21]. Six new CYP families were discov- one synthesis [19,23]. Specifically, disembodied ered; mitochondrial clan families CYP362 and 363, CYP2 (Cyp302A1; dib), shade (Cyp314A1, shd), and shadow clan families CYP364 and 370, and CYP3 clan families (Cyp315A1; sad) are all mitochondrial CYPs involved in CYP360 and 361 (Fig. 1, 2). the last three steps of 20-hydroxyecdysone (20-HE) syn- thesis. Cyp314A1 is the CYP required for the conversion Phylogenetic tree of to its active form, 20-HE [19,23]. The other Bayesian inference and maximum parsimony yielded three CYPs are divided into two new families CYP362 nearly identical topologies, so we have presented the tree (Cyp362A1, Cyp362A2) and Cyp363A1, indicating that in which branch lengths are proportional to the amount these CYPs are not as well conserved and probably have of change occurring in each lineage (Fig. 2). This tree is taken on new roles. A tree of the mitochondrial CYP clan also available in a detailed, expandable, readable pdf doc- is available as supplementary material (Additional file 4). ument as supplementary material (Additional file 2). The Bayesian tree includes posterior probabilities at nodes The CYP2 clan of D. pulex contains 21 members, includ- which reflect the proportion of trees sampled during the ing one pseudogene (not shown in Fig. 2, 3), separated search that included each particular node. The sea anem- into five distinct families (Cyp18, 306, 307, 364, 370) and one genome has not been manually curated and therefore six subfamilies. Two of the genes, phantom (Cyp306A1; the data are subject to potential assembly errors until val- phm) and spook (Cyp307A1; spo) are conserved Hallow- idated. However, the sea anemone provides an ancient een genes involved in the early stages of ecdysone synthe- anthozoan class cnidarian, and more importantly a sis [19,24]. The exact role of Cyp307A1 in ecdysone diploblast, to the analysis as the only non-triploblastic biosynthesis is unknown, but appears to be part of the species in our phylogenetic tree. The honeybee and fruitfly "Black box" of reactions involved in the earlier steps of provide a protostome insect CYP genome for comparison, ecdysone synthesis from cholesterol. Daphnia pulex only while the human CYPs provide a deuterostome for com- contains one Cyp307 gene similar to sequenced lepidop- parison to the Branchiopod crustacean, D. pulex. terans, but different than Drosophila that use two Cyp307 genes at different stages of their lifecycle [24,25]. Interest- In general, the tree shows six distinct monophyletic ingly, the Cyp307 genes are a sister group to the non- clades, all but one supported by posterior probabilities of CYP2 members (Fig. 3). 1.00. These include the mitochondrial, CYP2, CYP3, and CYP4 clans, and two deep branches that do not include The other three CYP2-clan families in D. pulex (Cyp18, any arthropod CYPs. The non-arthropod lineages are 364, 370) are divided into four subfamilies and contain nearly deuterostome exclusive and would be if not for the 19 genes. Interestingly, a sister-group in the CYP2 clan two anemone CYPs that show their closest relationships contains no arthropod CYPs. This group primarily con- to CYP26. These anemone CYPs found nested within a tains anemone CYPs, but also contains CYP17, CYP21, vertebrate lineage indicate that a CYP26-like ancestor may and the CYP1 family members inducible by chlorinated have existed in cnidarians but was lost in protostomes. hydrocarbons in vertebrates [26](Fig. 3; also available as Similar inferences concerning CYP loss in protostomes Additional file 5). The Cyp370 family is the largest of the have already been made about CYP51, CYP20 and possi- CYP2-clan families, containing 15 members with 13 bly CYP7. The history of CYP20 in protostomes may be members in the 370A subfamily and 2 members in the complex since an ortholog has been detected in the anne- 370B subfamily (Cyp370B1,2). There is also one pseudo- lid leech Haementeria depressa (CN807321). In addition, gene in the Cyp370A subfamily, Cyp370A3P. CYP19 (aromatase) clusters with the CYP2 and mitochon- drial clans, and CYP46 and related sea anemone CYPs are The Daphnia CYP370 family is greatly expanded relative part of a sister clade to the CYP4 clan (Additional file 2). to the single gene CYP15 and CYP303 families in insects it most closely resembles. Cyp15A1 is a regio- and stereo- Overall, the tree demonstrates that the four major clans specific epoxidase critical in the formation of juvenile hor- found in insects (mitochondrial, CYP2, 3, 4) encompass mone III (JH III) from methyl farnesoate in the corpora all of the CYPs in D. pulex. An Excel table of each of the allata of the cockroach [27]. However, methyl farnesoate, CYP genes and pseudogenes found in the Daphnia pulex a juvenile hormone precursor, is considered the major ter- genome, their nucleotide and amino acid sequences, and penoid hormone in [28]; therefore, a methyl links to their scaffold position is available as supplemen- farnesoate epoxidase is unnecessary and it is unlikely that tary material (Additional file 3). the CYP370A and CYP370B subfamily members specifi- cally perform this function. The role of the Cyp370 family The mitochondrial clan of D. pulex contains six members in Daphnia is currently unknown. Cyp18 and Cyp364 are in five families and five subfamilies. Three of the members both close relatives of Cyp306 (phantom), suggesting are highly conserved involved in ecdys- potential involvement in ecdysone synthesis or catabo-

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CYP71 A H

CYP81 A H

CYP8B1 H

CYP71 B H 1.00 CYP3A1 9 H CYP5 1 H CYP30A1 7 ³1RQ-DUWKURSRG´ CYP30A2 7

CYP370A4

CYP30A6 7

CYP30A7 7

CYP30A5 7 CYP30A8 7 Human CYP30A13 7

CYP30A9 7

CYP370A10

CYP30A12 7

CYP370A11

CYP30B1 7

CYP370B2

CYP1A1 5 B

CYP303A1 B

CYP303A1 M

CYP3 05D1 B

CYP35A1 0 M

CYP33A1 4 B

CYP3 42A1 B

CYP34A1 0 M

CYP18A1

CYP18A1 B

CYP1A1 8 M

CYP306A1 B

CYP306A1 M

CYP36A1 0

CYP26 A H

CYP27 A H

CYP2A13 H

CYP2 S1 H

CYP2B6 H

CYP21 F H

CYP29 C H

CYP219 C H

CYP218 C H

CYP28 C H

CYP21 E H

CYP26 D H

CYP2 W1 H

CYP22 J H

CYP2R1 H

CYP21 U H

CYP364A1

CYP34A2 6

CYP34A3 6

CYP1A1 H CYP12 A H CYP2 CYP11 B H

XM 001636548

XM 001629397

XM 001641370

1.00 XM 001624612

XM 001640787 XM 001638519 Daphnia XM 001641227

CYP1 7 H

CYP2A2 1 H

XM 001640204

XM 001629683

XM 001639012 XM 001638989 Honeybee XM 001638631

XM 001632943

XM 001622885

XM 016276780

XM 001627679

XM 001627677 XM 001633263 Drosophila

XM 001620496

XM 016332970

XM 001622925

XM 001622289

XM 001634749

XM 016386180

XM 001632313 Human

XM 001626823

XM 001640220

XM 001621453

XM 001631205

XM 001636040 XM 001636039 Anemone XM 001635327

XM 001635285

XM 001632195 0.89 XM 001632149 XM 016217460

CYP307A1

CYP307B1 B

CYP37A1 0 M

CYP32A1 6

CYP32A2 6

CYP334A1 B

CYP301A1 B

CYP31A1 0 M

CYP31B1 0 B

CYP4A1 9 M

CYP12A4 M

CYP12A5 M

CYP12C1 M

CYP12E1 M

CYP1D1 2 M

CYP1B2 2 M

CYP32A1 0 B

CYP302A1 M

CYP32A1 0

CYP34A1 1 B

CYP34A1 1 M

CYP314A1

CYP363A1

CYP35A1 1

CYP35A1 1 M

CYP35A1 1 B

CYP1 1B1 H CYP11B2 H Mitochondrial CYP1A1 1 H

CYP27B1 H

CYP27C1 H

CYP2A1 7 H

XM 001622478

XM 001622474 1.00 XM 001633936 Daphnia XM 001633907

XM 001627469

XM 001632334

XM 001640379

XM 001636532

XM 001636474 XM 001637421 Honeybee

CYP24 H CYP19 CYP1 9 H

CYP4C55

CYP456 C

CYP4C31

CYP433 C

CYP453 C Drosophila

CYP458 C

CYP4C59

CYP434 C

CYP4 C3 M

CYP4V2 H XM 001623827 Human XM 001623826

CYP4V1 A B

CYP4 AP3

CYP4P4 A

CYP4P5 A

CYP4P2 A CYP4 AP1 Anemone CYP4P8 A

CYP4AP7

CYP4P6 A

CYP4 AN4

CYP4N5 A

CYP4N2 A

CYP4N6 A

CYP4AN7

CYP4 AN8

CYP4 AN9

CYP4N1 A 0

CYP4N1 A 1

CYP4 BY1

CYP4Y2 B

CYP4Y3 B

CYP4Y5 B

CYP4Y6 B

CYP4 BY4

CYP4BX1

CYP4X4 B

CYP4X2 B

CYP4X3 B

CYP4 G11 B

CYP4 G15 M

CYP4 G1 M

CYP41 D M

CYP312A1 M

CYP4S3 M

CYP42 D M

CYP4D8 M

CYP414 D M

CYP420 D M CYP421 D M CYP4 CYP4E1 A M

CYP41 E M

CYP42 E M

CYP4E3 M

CYP4 AD1 M

CYP4AA1 B

CYP4AA1 M Daphnia

CYP41 P M

CYP43 P M

CYP42 P M

CYP4C1 A M

CYP4C2 A M CYP4AC3 M Honeybee CYP4B3 A B

CYP4A11 H

CYP422 A H

CYP41 X H

CYP41 Z H

CYP4B1 H CYP42 F H Drosophila CYP43 F H

CYP411 F H

CYP412 F H

CYP48 F H

CYP422 F H

XM 001639310

CYP33A2 1 M Human CYP33A3 1 M

CYP313A1 M

CYP313A5 M

CYP313A4 M

CYP313B1 M

1.00 CYP38A1 1 M

CYP31A1 1 M Anemone

CYP316A1 M

XM 001639142

XM 001634409

XM 001634408

XM 001630409

XM 001624280

XM 001626846

CYP4 6 H

XM 001624290

CYP30A1 6

CYP30A5 6

CYP360A4

CYP360A3

CYP3 60A6

CYP30A7 6

CYP3 60A10

CYP30A8 6

CYP30A9 6

CYP360A11

CYP34 A H

CYP3A7 H

CYP35 A H

CYP343 A H

CYP51 A H

XM 001638462

XM 001622177

XM 001630310

XM 001625609

XM 001625608

XM 016376660

XM 001627680

XM 016235580

XM 016223520

XM 016238470

XM 001622314

XM 001622313

XM 001628036

XM 001628017

CYP31A1 6

CYP31B1 6

CYP6 G1 M

CYP6V1 M

CYP6T1 M

CYP63 T M

1.00 CYP6 W1 M

CYP6 G2 M

CYP6Q1 A B

CYP6 BD1 B

CYP6AS1 B

CYP6AS2 B

CYP6AS13 B

CYP6S3 A B

CYP6R1 A B

CYP6AS5 B

CYP6AS12 B

CYP6S7 A B

CYP6S8 A B

CYP6AS11 B

CYP6AS15 B CYP6 AS14 B CYP3 CYP6AS10 B

CYP6S17 A B

CYP6AS18 B

CYP6S4 A B

CYP6C1 B B

CYP6E1 B B

CYP6 A9 M Daphnia

CYP621 A M

CYP62 A M

CYP68 A M

CYP618 A M

CYP617 A M

CYP623 A M

CYP6A19 M Honeybee

CYP37A1 1 M

CYP622 A M

CYP616 A M

CYP613 A M

CYP6D2 M CYP64 D M Drosophila CYP65 D M

CYP91 P B

CYP92 P B

CYP9 Q1 B

CYP9Q2 B

CYP9 Q3 B CYP9R1 B Human CYP91 S B

CYP9B1 M

CYP9B2 M

CYP9C1 M

CYP9H1 M

CYP9F2 M

CYP2D1 8 M Anemone

CYP2D2 8 M

CYP28A5 M 0.54 CYP2C1 8 M CYP39A1 0 M

CYP39A2 0 M

CYP36A1 3 B

CYP38A1 0 M

CYP61 U M

CYP3 10A1 M

CYP26B1 H

CYP26C1 H CYP2A1 6 H - 1.00 XM 001623765 ³1RQ DUWKURSRG´

XM 001636310

CYP20A1 H 0.1 changes Human, Anemone PhylogeneticFigure 2 relationship of the different CYP clans Phylogenetic relationship of the different CYP clans. Five CYP genomes were subjected to phylogenetic comparisons using MrBayes. An (H) after the CYP name denotes human sequences, a (B) denotes honeybee sequences, a (M) denotes Dro- sophila melanogaster sequences, CYP names lacking a letter are D. pulex sequences, and anemone sequences are noted with their GenBank protein accession numbers (start with XM). Numbers at nodes are posterior probabilities from the Bayesian analysis. Figure 2 is also available in an easily readable, expandable pdf format as a supplementary figure that allows recognition of individual CYP isoforms within each clade (Additional file 2).

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1.00 CYP370A1 CYP2 CY P370A2 1.00 CY P370A4 1.00 CY P370A6 0.92 CY P370A7 1.00 1.00 CY P370A5 CY P370A8 CYP 1.00 CY P370A13 1.00 CY P370A9 0.71 CY P370A10 370 1.00 CY P370A12 0.54 CY P370A11 1.00 CY P370B1 Daphnia 1.00 CY P370B2 0.97 CY P1 5A 1 B 1.00 CY P303A1 B 0.55 CY P303A1 M 1.00 1.00 CY P3 05 D1 B 1.00 CY P305A1 M CY P343A1 B 1.00 CY P342A1 B CY P304A1 M 1.00 CY P18A 1 CYP2 1.00 CY P1 8A 1 B 1.00 CY P1 8A 1 M 1.00 CY P306A1 B Daphnia 1.00 1.00 CY P306A1 M CY P306A1 Honeybee CY P2A6 H 0.77 1.00 CY P2A7 H CY P2A13 H Drosophila 1.00 CY P2S1 H CY P2B6 H Human 1.00 CY P2F1 H CY P2 C9 H 0.99 0.64 CY P2 C1 9 H 0.53 0.72 1.00 CY P2 C1 8 H 1.00 CY P2 C8 H CY P2E1 H 1.00 0.62 CY P2 D6 H CY P2W1 H 1.00 CY P2J2 H CY P2 R1 H 0.99 CY P2 U1 H 1.00 CY P364A1 1.00 CY P364A2 CY P364A3 1.00 CY P1A1 H 1.00 CY P1A2 H 0.67 CY P1B1 H XM 00 16 36 54 8 0.92 1.00 XM 00 16 29 39 7 1.00 1.00 XM 00 16 41 37 0 XM 00 16 24 61 2 1.00 XM 00 16 40 78 7 1.00 XM 00 16 38 51 9 0.70 XM 00 16 41 22 7 1.00 CY P17 H CY P2 1A 2 H XM 00 16 40 20 4 XM 00 16 29 68 3 1.00 XM 00 16 39 01 2 0.94 XM 00 16 38 98 9 XM 00 16 38 63 1 1.00 0.98 0.80 0.61 XM 00 16 32 94 3 XM 00 16 22 88 5 XM 00 16 27 67 8 0.98 XM 00 16 27 67 9 CYP2 XM 00 16 27 67 7 1.00 XM 00 16 33 26 3 1.00 XM 00 16 20 49 6 Human 1.00 XM 00 16 33 29 7 1.00 1.00 XM 00 16 22 92 5 0.64 XM 00 16 22 28 9 Anemone XM 00 16 34 74 9 XM 00 16 38 61 8 1.00 XM 00 16 32 31 3 1.00 XM 00 16 26 82 3 0.99 1.00 XM 00 16 40 22 0 1.00 XM 00 16 21 45 3 XM 00 16 31 20 5 1.00 XM 00 16 36 04 0 1.00 XM 00 16 36 03 9 1.00 XM 00 16 35 32 7 1.00 XM 00 16 35 28 5 CYP307 1.00 XM 00 16 32 19 5 XM 00 16 32 14 9 0.99 XM 00 16 21 74 6 Daphnia 0.98 CY P307A1 1.00 CY P307B1 B Honeybee CY P307A1 M Drosophila

PhylogeneticFigure 3 relationship of the different CYP2 clan members Phylogenetic relationship of the different CYP2 clan members. Three CYP2 clades are observed; two that contain arthropod CYP2 members and one that does not. In addition, the CYP2 clan is expanded in Daphnia pulex because of expan- sion of the Cyp370 family. An (H) after the CYP name denotes human sequences, a (B) denotes honeybee sequences, a (M) denotes fruitfly sequences, CYP names lacking a letter are D. pulex sequences, and anemone sequences are noted with their GenBank protein accession numbers (start with XM). Numbers at nodes are posterior probabilities.

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lism (Fig. 3). The Cyp364 family is a new family that con- able on GenBank (BQ703381 and BQ703379, respec- tains three genes (Cyp364A1,2,3). Cyp18, which is also tively). The D. pulex genome sequence coverage was 8.7×, found in insects, is induced by 20-HE in Drosophila [29]. therefore our inability to find these two CYPs may be due to the known gaps in the genome assembly. It is also pos- The CYP3 clan consists of numerous CYPs involved in sible that these two genes were deleted from the Daphnia detoxification of xenobiotics and endobiotics [30-33]. Genomics Consortium's chosen parthenogenic D. pulex or Some CYP3 clan members are inducible by hormones "chosen one" due to strain differences. such as progesterone [34] and ecdysone [17], and are responsible for the metabolism and elimination of The Cyp4 members are considered the least studied of the hormones in vertebrates [20,35,36]. Although the poste- CYP clans in insects [22] and are involved in fatty acid rior probability at the base of the CYP3 clan is low (0.54), metabolism, including inflammatory arachidonic acid this only reflects the uncertainly of the position of the first metabolites, and xenobiotic metabolism in mammals two Drosophila lineages at the base of the CYP3 clan. The [37]. Some CYP4 members may be involved in sensory CYP3 clade is strongly supported when Drosophila is not perception in insects as they are found in the included in the analysis (1.00). In Daphnia pulex, the [38]. A Cyp4c member is also involved in the biosynthesis CYP3 clan contains 12 genes and one pseudogene, of juvenile hormone, and another is inducible by hyper- arranged into two new families (Cyp360, Cyp361) and trehalosemic hormone, a key hormone in arthropod car- three subfamilies. Eleven of these thirteen genes are in the bohydrate metabolism [39]. Furthermore, some Cyp4 Cyp360A subfamily leaving just Cyp361A1 and members are down-regulated by ecdysteroids [40], indi- Cyp361B1 outside this subfamily in the Cyp3 clan of D. cating that Cyp4 members may play a key role in sensory pulex. The closest relatives of the Cyp360 subfamily in the and hormonal functions in D. pulex. tree are the Cyp6 and Cyp9 subfamily members of insects involved in endobiotic and xenobiotic metabolism and Primarily CYP3, but some CYP4 and mitochondrial clan detoxification. Similarly, the closest relatives of the two members have been associated with resistance to pesti- Cyp361 subfamily members are the anemone CYP3-like cides [17,30,41-44]. Several Cyp3 clan members, such as group, and the human CYP3A and CYP5A subfamily Cyp6g1 and Cyp6a5 are associated with DDT or pyre- members involved in detoxification and thromboxane A2 throid resistance, respectively [41,44]. Cyp4D10 and biosynthesis. In general, the CYP3 clan in insect species other CYP4 members in Drosophila are inducible by plant has more CYPs than the Daphnia pulex CYP3 clan. Most alkaloids and may be important in plant host interactions CYP families in insects have only a few members, but the [45]. In D. pulex, differential expression of two Cyp4 genes CYP6AS subfamily has 18 members, 37.5% of the honey- is associated with resistance to tannic acid and leaf litter bee P450s, and there are 35 members of the CYP3 clan [15]. Cyp4C32 expression is much higher in ecotype 1 distributed between seven families making up 42% of the and Cyp4AP1 shows much higher levels of transcription D. melanogaster P450s. The CYP3 clan in insects, and in in ecotype 2, which is exposed to high amounts of leaf lit- particular the CYP6AS subfamily in honeybee was ter and polyphenols, and in turn resistant to toxic leaf lit- recruited for major gene expansion as were the CYP360 ters [15]. Interestingly, the CYPs that show differential and CYP370 families in D. pulex, and to a lesser degree the expression based on ecotype and leaf litter exposure, are CYP4C subfamily. A tree of the CYP3 clan is available as the two CYPs that are not found within the D. pulex v1.1 an additional file (Additional file 6). genome sequence assembly. The complete sequence of these two CYPs is not available [15]. The CYP4 clan, which is the sister-group to a clade con- taining the CYP3 clan plus one of the two "non-arthro- Expansion of the CYP 2 and 4 clans pod" clans, consists of 38 members all in the same family A comparison of the number of genes in each of the CYP (Cyp4) and arranged into five subfamilies (Cyp4C, clans from D. pulex, silkmoth (Bombyx mori), human Cyp4AN, Cyp4AP, Cyp4BX, Cyp4BY) with 4–10 members (Homo sapiens), pufferfish (Fugu rubripes), honeybee in each subfamily (Additional file 7). There is also a pseu- (Apis mellifera), sea urchin (Stronglyocentrotus purpuratus), dogene in the Cyp4C subfamily. Two members of the and fruitfly (Drosophila melanogaster) indicates both subtle Cyp4 family were not observed in the Daphnia pulex and demonstrative differences in CYP clan numbers genome v1.1 draft genome sequence assembly (Septem- between species (Fig. 4). In general, the protostomes ber, 2006), but were cloned by degenerative PCR in a pre- (fruitfly, honeybee, D. pulex) have significantly fewer vious study in which nine CYP4 members were partially CYP2 clan members than deuterostomes, indicating an cloned [15]. The two absent CYPs, Cyp4C32 (95% identi- expansion of the CYP2 clan in deuterostomes (Fig. 3). Of cal to 4C34v1; 89% to 4C34v2 from the Daphnia pulex the protostomes investigated, D. pulex has the greatest per- genome) and Cyp4AN1 (92% to 4AN2v1; 96% identical centage of CYP2 clan members. The CYP2 clan encom- to CYP4AN2v2 from the Daphnia pulex genome), are avail- passes approximately 5.5–10% of the total CYPs in most

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which is phylogenetically related to the CYP15 and CYP303 families, has expanded dramatically in D. pulex, and this family lacks a specific enzyme with a known func- tion. Based on our current knowledge of CYPs, the expan- sion of the CYP370 family is probably necessary for responses to environmental stressors such as toxicants,

and/or other growth or behavioral stimulators such as

¡ ¢ £ ¤ ¥ ¦ ¡ ¢ plant alkaloid toxins.

Tandem repeat regions Tandem duplicates are genes that are within intron dis- tances of each other, nearly identical (95+% identity), and may harbor some interesting biology. Gene expansion by tandem duplication is common in P450 evolution but the

basis for recruitment of a founder gene is not understood.

§ §

¡ ¡ ¤ There are several CYP tandem repeat regions in the D. pulex genome (Fig. 5). Overall, 45 of the 77 (58%) CYP Comparisonfrommoth,Figure Daphnia, Drosophila, 4 of Honeybee,the andnumber Human Sea of ge Urchgenomesnesin, in Fugueach (pufferfish),of the CYP clansSilk- genes are located within tandem repeats. Twenty-eight of Comparison of the number of genes in each of the the 37 (76%) CYP4 family members, including Cyp4C53, CYP clans from Daphnia, Honeybee, Sea Urchin, are found in tandem repeat regions, which explain in part Fugu (pufferfish), Silkmoth, Drosophila, and Human the expansion of the CYP4 clan in D. pulex. In contrast, the genomes. The data was derived from [22,46-49] and manual CYP3 clan has 69% (9/13) of its genes found in tandem curation of the Daphnia pulex genome. repeat regions, including 9/11 of the Cyp360 family members. However, the CYP3 clan is small relative to the insect genomes, thus presence in tandem repeats is not insects with honeybee the exception at 17.4% of its CYPs correlated to the size of the clan. Lastly, none of the mito- in the CYP2 clan [22]. In contrast, D. pulex has 21 CYPs in chondrial CYPs, many of which have specific functions in the CYP2 clan, nearly double of any sequenced insect (11/ ecdysone synthesis [19], are members of a tandem repeat 132 in Aedes aegypti), and D. pulex's CYP2 clan members region (Fig. 4). Expansions of conserved endogenous encompass 26.9% of its CYPs, indicating a significant functions are rare and may be selected against. Con- expansion of this clan relative to the insects. No other versely, tandem duplications may indicate diversity in crustacean has been sequenced, so whether the expansion exogenous xenobiotic substrates [3]. of the CYP2 clan is typical of crustaceans is unknown. The CYP2 clan is highly expanded relative to the insects, The CYP4 clan is also slightly expanded in D. pulex relative but only 9 of the 21 CYP2 (43%) clan genes are members to the insects. There are 38 CYP4 members that encom- of a tandem repeat region. Initially, we thought this indi- pass 49% of the CYPs in the genome. The CYP4 clan varies cated that tandem repeat regions had little to do with the from 8.6–42% of the CYP genome in sequenced insects expansion of the CYP2 clan in D. pulex. However, all nine [22]. Excluding the honeybee, which only has 4 CYP4 of the tandemly repeated CYP2 clan members are in the members, the rest of the insect's CYP4 members vary from CYP370 family. It is interesting that the rest of the CYP2 30.7–42% of the total CYPs. Relative increases in CYP2 clan members are not found in tandem repeat regions, and CYP4 members in D. pulex leaves a relative reduction and it is tempting to speculate that most D. pulex CYP2 in the CYP3 clan compared to insects. Only 16.7% (13/ clan members, just as many of the mitochondrial CYPs, 78) of the members of the D. pulex CYP genome are CYP3 have highly specific functions such as ecdysone biosyn- clan members; whereas 38–61% (28–76) of the insect thesis. Needs for P450s are often met by expansion via CYPs are CYP3 clan members. tandem duplication leading to gene clusters. This suggests that the CYP370 family expanded while under selective There are several CYP2-clan members similar in structure pressure. Which genes expand may be independent of to other ecdysone metabolizers (CYP18, CYP364 mem- clan or family membership and depend on substrate spe- bers). However, the formation of juvenile hormone III cificity required to cope with a new xenobiotic stress. The from methyl farnesoate by CYP15A1 is unnecessary in C. elegans genome has almost half of its P450s in the CYP2 crustaceans and D. pulex in turn lacks the CYP15A1 gene. clan, yet it only has one mitochondrial clan member, In addition, D. pulex lacks the CYP303 subfamily mem- CYP44. Insects have expanded the CYP3 clan into the bers with unknown but putative external sensory develop- large CYP6 and CYP9 families and several spinoff fami- ment function [5]. Nevertheless, the CYP370 family, lies. Deuterostomes have expanded CYP2 extensively,

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TheFigure Cytochrome 5 P450s are often found in tandem repeats on the genome The Cytochrome P450s are often found in tandem repeats on the genome. Forty-five of the seventy-seven discov- ered CYPs are found on tandem repeat regions, and an additional CYP (Cyp4C53; 1481785) is found adjacent to the Cyp4C- rich region on scaffold 5 (not shown). The scaffold numbers are presented on the left hand side of the figure and the positions of each tandem repeat on the scaffold are presented before and after each break in the tandem repeat region. Arrows indicate the direction of the repeat regions, and the names of each CYP are provided above each block that represents a single CYP gene.

while Trichoplax adhaeren has only one CYP2 clan member Cyp4BY5 gene provided no evidence of this and this CYP (e_gw1.8.275.1|Triad1 at JGI). firmly fits in the Cyp4BY subfamily based on identity and phylogenetic status (Fig. 2). Scaffold 4 is especially rich in tandem repeat regions. It contains 26 of the 44 (59%) CYP genes located in tandem Conclusion repeats, and 19 of these genes are CYP4 family members. The cladoceran, D. pulex has a wide range of CYPs with the Scaffold 4 contains tandem repeats for CYP4AN, 4AP, same clans as insects, but with distinct changes in the pop- 4BX, and 4BY subfamilies. Interestingly, a Cyp4BY sub- ulation of each clan. Of note is the expansion of the newly family member (Cyp4BY5) is located in the middle of the discovered CYP370 family. Elucidation of the function of Cyp4AN subfamily tandem repeat region. This is unusual the different CYP families and subfamilies will no doubt as most of the genes adjacent to each other in a tandem provide important insight into the ability of D. pulex to repeat belong to the same subfamily. As the genes diver- respond to environmental changes, predator-prey rela- sify a single gene cluster may contain multiple subfamilies tionships, hormonal changes, plant allelochemicals, and as in the CYP2ABFGST and the CYP4ABXZ gene clusters in sensory stimuli. Ultimately, the understanding of the evo- mammals [46]. An error could have been made in the lution of animals will require deciphering the history of annotation of this CYP; however, re-examination of the P450s and the Daphnia genome may contribute to

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unraveling molecular aspects of the ecology and physiol- tution model. The parsimony tree was based on a heuristic ogy of this commonly used crustacean species. search with 10 random addition sequence replications and tree-bisection-reconnection branch swapping, with Methods gaps treated as missing data. A 50% majority-rule tree was Manual curation computed for all equally parsimonious trees. The Joint Genome Institute (JGI) http://www.jgi.doe.gov/ Daphnia/ and wFleaBase http://wfleabase.org generated Next, we constructed several trees using Bayesian infer- gene models for protein-coding genes in Daphnia pulex ence, a probabilistic model-based method of phylogeny using multiple algorithms. The models were constructed reconstruction that is similar to maximum likelihood but and filtered in order to reduce redundancy based on which has substantially reduced computation time. Baye- homology and EST support, which in turn produced the sian trees were constructed with MrBayes version 3.1.2 Dappu v1.1 gene builds (July, 2007). The gene models [52] on a computing cluster provided by the Computa- were directly compared to other genomes such as human, tional Biology Service Unit of Cornell University http:// mouse, Drosophila, zebrafish (Danio rerio), Xenopus, and cbsuapps.tc.cornell.edu/mrbayes.aspx. We built trees bovine (Bos taurus) on the Daphnia genome portal. We using the "mixed-model" approach in which the Markov searched for cytochrome P450 (CYP) gene models using chain Monte Carlo sampler explores nine different fixed- KEGG and KOG pathways in addition to the Advanced rate amino acid substitution models implemented in Search Options on the JGI Daphnia pulex portal, and then MrBayes. We used 4 chains with runs of 2.5 million gen- manually curated each of the CYPs with the help of the erations with chains sampled every 100 generations and gene models and genome comparisons detailed above. with a burnin of 10,000 trees; the WAG [53] model was Our manual annotations included corrections to several selected as the best fitting substitution model by MrBayes. models based on knowledge of intron-exon boundaries in Due to the difficulty in choosing an outgroup for such a related genes and BLAST searches as described previously diverse and ancient gene family, we elected to present [47], and later assigning gene names based on the homol- phylogenies with a midpoint rooting in which the root is ogy of Daphnia CYPs to other species using defined placed halfway between the two most divergent nomenclature and naming rules for complete genes and sequences. Midpoint rooting will accurately determine pseudogenes [21,46]. the root of a phylogeny provided that rates of substitution do not vary across the tree. To examine this assumption of Gross Comparisons and Phylogeny a molecular "clock", we repeated the Bayesian analyses The different CYP clans and families from Daphnia pulex with the added constraint of constant rates of amino acid were compared to CYP genomes from human (Homo sapi- substitution and rooted the resulting trees with a mid- ens), honeybee (Apis mellifera), fruitfly (Drosophila mela- point rooting. The "clock" constrained trees yielded the nogaster), silkmoth (Bombyx mori), purple sea urchin same overall topology and root position as the uncon- (Stronglyocentrotus purpuratus), and pufferfish (Fugu strained analyses, indicating that our application of a rubripes) [22,46-49]. In addition, phylogenetic compari- midpoint rooting is justified. sons of the different full length Daphnia pulex CYP genes were performed with full length human (Homo sapiens), Tandem repeat regions fruitfly (Drosophila melanogaster), and honeybee (Apis mel- Automated genome annotation software that relies on lifera), and select starlet sea anemone (Nematostella vecten- alignment with gapping, from BLAST and BLAT thru sis) CYPs. Honeybee, fruitfly, and human CYPs have been GeneWise, Exonerate and similar tools, may have prob- manually curated, but the anemone CYPs are only availa- lems identifying tandem genes and pseudogenes in areas ble through GenBank, http://www.stellabase.org, or the with highly identical exons [54]. Software may skip over Nelson cytochrome P450 webpage [49], and have not one exon and link to its nearly identical downstream gene been curated or officially named yet. This genome was model in tandem repeat regions. The Daphnia pulex chosen as an ancient, diploblast for comparison to proto- genome appears rich in tandem genes, and CYPs are often stome (honeybee, fruitfly, Daphnia) and deuterostome found in tandem repeat regions. Tandemgenes, or 'Tandy', (human) CYP genomes. software http://eugenes.org/gmod/tandy/ was used to address this problem, and potential CYPs found in tan- To construct phylogenetic trees, all of the Daphnia, dem repeat areas were provided by Don Gilbert, Biology, human, fruitfly, honeybee, and anemone CYP amino acid Indiana University http://wfleabase.org/genome-summa sequences were first aligned using default parameters in ries/gene-duplicates/tdpageCytochrome_P450_15.html. ClustalX [50]. Trees were constructed using two methods. Areas potentially containing tandem repeats were care- First, we used maximum parsimony as implemented in fully mined and manually curated using the newly gener- PAUP 4.0b10 [51], a method that minimizes the number ated models, and maps were made of each of the tandem of evolutionary events but does not use an explicit substi- repeat regions.

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Authors' contributions WSB conceived and coordinated the study, annotated the Additional file 6 CYPs, aligned the sequence data, and was the primary Phylogenetic tree of the CYP3 clan members. An (H) after the CYP author drafting the manuscript. PBM carried out the phy- name denotes human sequences, a (B) denotes honeybee sequences, a (M) denotes fruitfly sequences, CYP names lacking a letter are D. pulex logenetic analysis and helped draft the manuscript. DRN sequences, and anemone sequences are noted with their GenBank protein checked the CYP annotations, named the CYPs, provided accession numbers (start with XM). valuable direction, insight, and expertise on CYP evolu- Click here for file tion, and helped draft the manuscript. All authors read [http://www.biomedcentral.com/content/supplementary/1471- and approved the final manuscript. 2164-10-169-S6.pdf]

Additional material Additional file 7 Phylogenetic tree of the CYP4 clan members. An (H) after the CYP name denotes human sequences, a (B) denotes honeybee sequences, a Additional file 1 (M) denotes fruitfly sequences, CYP names lacking a letter are D. pulex sequences, and anemone sequences are noted with their GenBank protein Comparison of the number of functional CYP genes in different accession numbers (start with XM). genomes. A pdf table comparing the number of functional CYP genes Click here for file from several different organisms. [http://www.biomedcentral.com/content/supplementary/1471- Click here for file 2164-10-169-S7.pdf] [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-169-S1.pdf]

Additional file 2 Phylogenetic relationship of the different CYP clans. Five CYP genomes Acknowledgements were subjected to phylogenetic comparisons using MrBayes. An (H) after The sequencing and portions of the analyses were performed at the DOE the CYP name denotes human sequences, a (B) denotes honeybee Joint Genome Institute under the auspices of the U.S. Department of sequences, a (M) denotes fruitfly sequences, CYP names lacking a letter Energy's Office of Science, Biological and Environmental Research Program, are D. pulex sequences, and anemone sequences are noted with their and by the University of California, Lawrence Livermore National Labora- GenBank protein accession numbers (start with XM). tory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Click here for file Laboratory under Contract No. DE-AC02-05CH11231, Los Alamos [http://www.biomedcentral.com/content/supplementary/1471- National Laboratory under Contract No. W-7405-ENG-36 and in collabo- 2164-10-169-S2.pdf] ration with the Daphnia Genomics Consortium (DGC) http://daph nia.cgb.indiana.edu. Additional analyses were performed by wFleaBase, Additional file 3 developed at the Genome Informatics Lab of Indiana University with sup- Detailed summary of each CYP sequence, its position on the D. pulex port to Don Gilbert from the National Science Foundation and the genome, and other potentially helpful information. An Excel workbook National Institutes of Health. Coordination infrastructure for the DGC is (xlsx) table of each of the CYP genes and pseudogenes found in the Daph- provided by The Center for Genomics and Bioinformatics at Indiana Uni- nia pulex genome, their nucleotide and amino acid sequences, links to versity, which is supported in part by the METACyt Initiative of Indiana Uni- their scaffold position, manual protein identifying number, and comments versity, funded in part through a major grant from the Lilly Endowment, Inc. about the annotation and/or presence of an expressed sequence tag (EST). Our work benefits from, and contributes to the Daphnia Genomics Con- Click here for file sortium. This is Technical Contribution No. 5608 of the Clemson Univer- [http://www.biomedcentral.com/content/supplementary/1471- sity Experiment Station, supported by the CSREES/USDA (project number 2164-10-169-S3.xls] SC-1700342).

Additional file 4 References Phylogenetic tree of the mitochondrial CYP clan members. An (H) 1. Wei P, Zhang J, Egan-Hafley M, Liang S, Moore DD: The nuclear after the CYP name denotes human sequences, a (B) denotes honeybee receptor CAR mediates specific xenobiotic induction of drug sequences, a (M) denotes fruitfly sequences, CYP names lacking a letter metabolism. Nature 2000, 407:920-923. are D. pulex sequences, and anemone sequences are noted with their 2. Kang HS, Angers M, Beak JY, Wu X, Gimble JM, Wada T, Xie W, Col- GenBank protein accession numbers (start with XM). lins JB, Grissom SF, Jetten AM: Gene expression profiling reveals Click here for file a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism. Physiol Genomics 2007, 31:281-294. [http://www.biomedcentral.com/content/supplementary/1471- 3. Li X, Schuler MA, Berenbaum MR: Molecular mechanisms of 2164-10-169-S4.pdf] metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol 2007, 52:231-253. Additional file 5 4. Estabrook RW: A passion for P450s (rememberances of the early history of research on cytochrome P450). Drug Metab Phylogenetic tree of the CYP2 clan members. An (H) after the CYP Dispos 2003, 31:1461-1473. name denotes human sequences, a (B) denotes honeybee sequences, a 5. Willingham AT, Keil T: A tissue specific cytochrome P450 (M) denotes fruitfly sequences, CYP names lacking a letter are D. pulex required for the structure and function of Drosophila sensory sequences, and anemone sequences are noted with their GenBank protein organs. Mech Dev 2004, 121:1289-1297. accession numbers (start with XM). 6. Waxman DJ: Interactions of hepatic cytochromes P-450 with steroid hormones: Regioselectivity and stereoselectivity of Click here for file steroid metabolism and hormonal regulation of rat P-450 [http://www.biomedcentral.com/content/supplementary/1471- enzyme expression. Biochem Pharmcol 1988, 37:71-84. 2164-10-169-S5.pdf]

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