Identification and evolution of nuclear receptors in Platyhelminths

Since the first complete set of Platyhelminth nuclear receptors (NRs) from Schistosoma mansoni were identified a decade ago, more flatworm genome data is available to identify their NR complement and to analyze the evolutionary relationship of Platyhelminth NRs. NRs are important transcriptional modulators that regulate development, differentiation and reproduction of animals. In this study, NRs are identified in genome databases of thirty-three species including in all Platyhelminth classes (Rhabditophora, Monogenea, Cestoda and Trematoda). Phylogenetic analysis shows that NRs in Platyhelminths follow two different evolutionary lineages: 1) NRs in a free-living freshwater flatworm (Schmidtea mediterranea) and all parasitic flatworms share the same evolutionary lineage with extensive gene loss. 2) NRs in a free-living intertidal zone flatworm (Macrostomum lignano) follow a different evolutionary lineage with a feature of multiple gene duplication and gene divergence. The DNA binding domain (DBD) is the most conserved region in NRs which contains two C4-type zinc finger motifs. A novel zinc finger motif is identified in parasitic flatworm NRs: the second zinc finger of parasitic Platyhelminth HR96b possesses a CHC2 motif which is not found in NRs of all other animals studied to date. In this study, novel NRs (members of NR subfamily 3 and 6) are identified in flatworms, this result demonstrates that members of all six classical NR subfamilies are present in the Platyhelminth phylum. NR gene duplication, loss and divergence in Platyhelminths are analyzed along with the evolutionary relationship of Platyhelminth NRs.


Introduction
Platyhelminths (flatworms) are one of the largest animal phyla, which includes more than 20,000 species [1,2]. Some flatworms are important disease-causing parasites of humans and livestock, e.g. Schistosomiasis, Paragonimiasis and Cestodiasis. Platyhelminths are bilaterally symmetrical, non-segmented acoelomates without an anus. Although they have an excretory system, they lack respiratory and circulatory systems. In addition, all flatworms are hermaphroditic, undergoing both asexual and sexual reproduction, with the exception of members in the Schistosomatoidae [3]. Platyhelminths are traditionally divided into four classes: Rhabditophora, Monogenea, Cestoda (tapeworms) and Trematoda (flukes). The class Rhabditophora includes all free-living flatworms, while all members in classes of Monogenea, Trematoda, and Cestoda are parasitic flatworms. From an evolutionary point of view, the parasitic classes arose from a primitive free-living flatworm [3]. Nuclear receptors (NRs) are important transcriptional modulators that regulate development, differentiation and reproduction of animals. Most of NRs share a common tertiary structure: A/B-C-D-E domains. The A/B domain is highly variable, the C domain is the DNAbinding domain (DBD) which is the most conserved region containing two zinc finger motifs, the D domain a flexible hinge between the C and E domains and is poorly conserved, the E domain contains the ligand binding domain (LBD) which is involved in transcriptional activation. Atypical NRs are found in some animals, e.g. NRs with a DBD but no LBD are found in arthropods and nematodes, members without a DBD but with a LBD are present in some vertebrates, and NRs with two DBDs and a single LBD (2DBD-NRs) are identified in protostomes. A phylogenetic analysis of the NRs divides them into six classical subfamilies by alignment of the conserved DBD [4]. The early study of the complete genome sequence of ecdysozoan Arthropods (Drosophila melanogaster) [5] the mosquito (Anopheles gambiae) [6], free-living nematodes (Caenorhabditis elegans and C. briggsae) [7,8], tunicata (Ciona intestinalis) [9], mammalians: rat (Rattus norvegicus), mouse (Mus musculus) [10] and human (Homo sapiens) [11]) revealed that NRs in insects, tunicata and mammalians share the same evolutionary lineage with extensive gene loss/duplication, while NRs in nematodes follow a different evolutionary lineage with a feature of multiple duplication of SupNRs and gene loss.
Since we identified the first complete set of Platyhelminths nuclear receptors (NRs) from Schistosoma mansoni a decade ago [12], more flatworm genome data has become available to identify their NR complement. Analysis of the NR complement of the blood fluke Schistosoma mansoni [12][13][14] and tapeworm Echinococcus multilocularis [13][14][15] shows that NRs in S. mansoni and E. multilocularis share the same evolutionary lineage as that of Deuterostomia and the arthropods in the Ecdysozoan clade of the Protostomia, but some divergent NRs in flatworms are not present in Deuterostomia or/and arthropods, for example 2DBD-NRs [16]. In this study, we analyzed genome databases of Platyhelminth Wormbase ParaSite including species from all of the four classes in Platyhelminths [17,18]. Identification of the NR complement will contribute to a better understanding of the evolution of NRs in Platyhelminths.
A schematic that illustrates the different NR subfamilies and NRs in human, insects, Mollusca and Platyhelminths are shown in Fig 1.

Data mining
Nuclear receptors in Platyhelminths were mined from the genome databases in Wormbase ParaSite (version: WBPS15 (WS276)) [17,18]. Amino acid sequence of DBD of SmTRa (AY395038), SmHR96b (AY688259) and Sm2DBDα (AH013462) were used as the query to tblastn (with E-value threshold: 1e-1) against all available databases. Any sequence that contains a zinc finger structure of the NR DBD (Cys-X2-Cys-X13-Cys-X2-Cys or Cys-X5-Cys-X9-Cys-X2-Cys) was retained, and the deduced amino acid sequence of the DBD was obtained by the conserved junction position (JP) and GT-AG rule if there was an intron in the DBD coding region. The analyzed Platyhelminth genome databases are listed in Table 1. The classification of Platyhelminths and a short description of each Platyhelminth species can be found in Table 1.

Phylogenetic analysis
Phylogenetic trees were constructed from deduced amino acid sequences of the DBD and/or ligand binding domain (LBD), the sequences are aligned with ClustalW [36], phylogenetic analysis of the data set is carried out using Bayesian inference (MrBAYES v3.1.1) [37] with a mix amino acid replacement model + gamma rates. The trees were started randomly; four simultaneous Markov chains were run for 5 million generations, the trees were sampled every 100 generations, the Bayesian posterior probabilities (BPPs) were calculated using a Markov chain Monte Carlo (MCMC) sampling approach implemented in MrBAYES v3.1.1, with a burn-in value setting at 12,500. The same data set was also tested by Maximum Likelihood method using PhyML 3.0 [38] with support values obtained by bootstrapping a 1000 replicates. The most optimal evolutionary models for phylogenetic reconstruction of NRs in each data set were determined by AIC criteria with Smart Model Selection in PhyML (SMS) [39] and the tested model for each data set was shown in each figure legend. For GenBank accession numbers of published NR sequences used in this study see S1 File.   [40,41] Free-living, intertidal flatworm that can regenerate most of its body parts.

Polyopisthocotylea Polystomatidae
Protopolystoma xenopodis (Px) [46] Fluke of African clawed frogs where the adult worms live in the host's urinary bladder.

NR complements in Platyhelminths
NRs are identified in the genomes of 33 Platyhelminth species including 2 species (Schistosoma mansoni and Echinococcus multilocularis) that have had their NR complement reported [12,15]. The analyzed flatworm species shown in Table 1

Phylogenetic analysis of NRs
Phylogenetic analysis of NRs using DBD sequences were carried out with Bayesian inference and Maximum Likelihood method. The DBD sequences of NRs from human, Drosophila and other animals were used as controls. Both methods place control NRs in mono groups and in correct families. Comparison of Bayesian inference and Maximum Likelihood method indicates Bayesian inference highly supports the analysis but Maximum Likelihood sometimes shows a lower support value (S3 File). This result indicates that Bayesian inference supports phylogenetic analysis of NRs using only DBD sequences. The amino acid sequence of the P-box in the DBD is unique to NR members, groups or subfamilies. This sequence is helpful for identification of NRs when the phylogenetic analysis support value is low. For example, P-box sequence ESCKG is unique to NR subfamily 5 members (FTZ-F1 and DHR 39 orthologues). In this study, phylogenetic analysis of G. salaris NRs shows a lower support value for GsFTZ-F1 in subfamily 5 (S3 File), but the P-box sequence of GsFTZ-F1 (ESCKG) clearly demonstrated that it belongs to subfamily 5. The P-box sequence of all analyzed Platyhelminths NRs were checked to make sure that they share the same sequence with that of same group members. NRs in each Platyhelminth species are phylogenetic analyzed and the phylogenetic trees are reconstructed (S1-S17 Figs). NRs in Platyhelminths are summarized in Table 2.

A novel zinc finger motif (CHC2) exists in DBD of parasitic Platyhelminth NRs
DBD is the most conserved region in NRs, it contains two C4-type zinc finger motifs. In each motif, four cysteine residues chelate one Zn 2+ ion. The first zinc finger (CI) contains a sequence element, the P-box [62,63] which is responsible for binding the target gene, and the second zinc finger (CII) contains a sequence element the D-box which is responsible for dimerization [62]. Previously, we isolated a partial cDNA of a trematode Schistosoma mansoni HR96b (SmNR96b) [12], unlike other NRs, SmNR96b contains a long amino acid sequence in the DBD with two introns located in this region. DBD of SmHR96b has a histidine residue replaced by a second cysteine residue in the D-Box in CII and it forms a novel zinc finger motif (CHC2). Recently, CHC2 zinc finger motif has been demonstrated to be present in HR96b of Cestoda and other Trematoda species [64,65]. In this study, we determined that all SmHR96b orthologues in parasitic Platyhelminths contain this novel CHC2 zinc finger motif (Fig 2). Thus, CHC2 type zinc finger motif represents a novel NR CII which has diverged in ancient HR96b in a common ancestor of parasitic Platyhelminths. The function of this novel CHC2 type motif in NRs is unknown. Recent study shows that SmHR96b (named Vitellogenic Factor 1 in [64,65]) is essential for female sexual development.

New NRs identified in Platyhelminths
Previously, we demonstrated that a novel NR2E member (orthologue of nematode Caenorhabditis elegans NHR236) was present in Cnidaria, Arthropoda, free-living Platyhelminths, Mollusca and Echinodermata [14]. In this study, an orthologue of NHR236 was identified in P. xenopodis (Table 2 and S3 Fig). Amino acid sequence alignment shows that NHR236 orthologues have a unique P-box sequence (CDGCRG) and they form a new NR subgroup in NR2E super group and its progenitor gene was present in a common ancestor of Porifera and Bilateral [14]. It is the first time that an orthologue of NHR236 has been shown to exist in parasitic Platyhelminths.
Two divergent NRs from subfamily 1 are identified in different species of Platyhelminths. Phylogenetic analysis shows that Platyhelminths NR1a is an orthologue of Schistosoma mansoni NR1 (SmNR1) [44], which is present in different species of the four Platyhelminth classes. The other NR1 divergent member (NR1b) is an orthologue of the Cestode Echinococcus granulosus HR3 (EgHR3) [45], it is present in all analyzed tapeworms and also exists in the free-living Platyhelminth S. mediterranea (nhr10, [46]) (S18 Fig and Table 2). Phylogenetic analysis of the DBD sequences shows that Platyhelminth NR1b group clustered together with Drosophila HR3/human ROR/Mollusca HR3 group,but is outside of the HR3/ROR group (S18 Fig). To further demonstrate whether Platyhelminth NR1b is an orthologue of HR3/ROR, DBD with LBD sequences or only LBD sequence of NR1b orthologue (EgHR3 and SmeNR1b) were analyzed. Phylogenetic analysis shows that EgHR3 and SmeNR1b are clustered with the E75 group if both DBD and LBD sequences are analyzed, but they are clustered with E78 group if only LBD sequences are analyzed. These results suggest that the orthologues of Platyhelminths

PLOS ONE
NR1b are a group of divergent NRs that were present in a common Platyhelminth ancestor (S19 and S20 Figs).

NR genes lost in Platyhelminths
Comparison of NRs in Platyhelminths, orthologues of RAR, PPAR, E75, ROR, ER, MR, PR, GR, AR, Knir and NR7/8 (a new identified subfamily [26,29]) are missing in all analyzed Platyhelminth species. Fax-1 is only identified in Rhabditophora M. lignano, it was lost in Rhabditophora S. mediterranea and all analyzed parasitic Platyhelminths and NHR236 orthologue is not identified in Cestoda and trematode species. In Cestoda, RXR and PNR are present in Diphyllobothriidae Order but they are missing in Cyclophyllidea Order. NR gene lost in Platyhelminths is shown in Table 2.

Divergent NRs
Divergent NR refers to a NR which has a typical P-box sequence in the DBD but does not fall into any 'typical' NR groups, for example Platyhelminths NR1a and NR1b with a typical Pbox of 'CEGCKGFFRR' belonging to NR subfamily 1 but they do not fit into any groups within the NR1 subfamily. It also refers to a NR which has an atypical P-box sequence in the DBD and does not fall into the present NR nomenclature [66].
Divergent NRs exist in various lineages of Platyhelminths. One divergent NR with a typical P-box (CEGCKGFFKR) which is the same as that of RXR/TR4/NR4A is found in Rhabditophora S. mediterranea and in Cestoda Mesocestoides corti. Most of the Platyhelminth divergent NRs possess an 'atypical' P-box sequence. For example, a NR with a P-box of 'CEPCKVFFKR' is identified in Monogenea G. salaris, a NR with a P-box of 'CEACKAFFQQ' is found in all analyzed species of Cestoda Hymenolepis family, a NR which has a P-box of 'CDSCRAFFEM' exists in Cestoda Taenia family, a NR with a P-box of 'CEACKSFFKR' is found in Cestoda Diphyllobothriidea order and seventeen NRs with various 'atypical' P-box sequences were

NR gene duplication in Platyhelminths
3.7.1. TR. One TR is identified in Monogenea; two are identified in Rhabditophora M. lignano and three are identified in S. mediterranea; and two are identified in Cestoda and Trematoda, respectively. Our previous study showed that two TR homologues are present in Platyhelminths [12][13][14]34]. Phylogenetic analysis suggested that Platyhelminth TR gene duplicated after the split of the trematodes and the turbellarians [34], thus the paralogue of the trematode TR was not present in a turbellarian and in turn the paralogue of turbellarian TR was not present in trematode species. Phylogenetic analysis in this study shows that all TRs of parasitic Platyhelminths are clustered in a group, two M. lignano TRs are clustered in a group and three S. mediterranea TRs are clustered in another group. This result suggests that TRs duplicated independently in M. lignano, S. mediterranea and parasitic Platyhelminths. In parasitic Platyhelminth TR groups, trematode TRa (orthologues of Schistosoma TRa, SmTRa) are clustered together with those of Monogenea and Cestoda, but trematode TRb (orthologues of Schistosoma TRb, SmTRb) group only contain trematode TRs. This result suggested that one TR was present in a common ancestor of Platyhelminths and trematode TRa was an ancient TR gene. Phylogenetic analysis shows that the two Cestode M. corti TRs are clustered together  (Fig 4). HR96d group only contains Platyhelminth M. lignano HR96s, but each group of HR96a, HR96b and HR96c contains NR96s from all species of the four classes of Platyhelminths.
Phylogenetic analysis shows that the BPP support values for HR96b and HR96c nodes are lower than 0.8 (Fig 4). The amino acid sequences and intron position of DBD region of HR96s were further analyzed, the result shows the amino acid length is the same within the members each of group of HR96a, HR96c and HR96d, but different in the members among groups. With the exceptionof M. lignano HR96a members have no intron located in their DBD region and the intron positions of the DBD region is the same in the members of each group but is different among groups. Amino acid length of members and intron position are unusual in Platyhelminth HR96b, most which have an extra exon inserted between the P-box and Dbox region that resulted in 2 introns located in this region. Most important, DBD of all parasitic HR96b members have a histidine residue replaced by a second cysteine residue in the D-Box forming a novel zinc finger motif (CHC2). Phylogenetic analysis shows HR96d is an ancient genewhich is supported by sequence analysis, with only members of Platyhelminth HR96d sharing the same DBD amino acid length and intron position with that of Mollusca HR96s (Fig 5). For all sequence alignment of HR96s and intron position see S4 File. This result suggests that there were four HR96s in a common ancestor of Platyhelminths and the ancient HR96 gene (HR96d) was lost in Schmidtea mediterranea, Monogenea, Cestoda and Trematoda (Fig 4). HR96 genes were amplified in M. lignano and each of the four HR96 genes gave birth to a total of sixteen NR96 genes. 3.7.3. RXR. Two RXRs were identified in Rhabditophora, Monogenea, Cestoda and Trematoda, respectively. Phylogenetic analysis shows that parasitic flatworm RXRs are clustered in two different groups: RXR1 group (Schistosoma RXR1 orthologues) and RXR2 group (Schistosoma RXR2 orthologues). RXR2 group is clustered with RXRs of free-living flatworms, Drosophila, Mollusca and human, which suggests that parasitic Platyhelminth RXR2 is an orthologue of free-living flatworms, Drosophila, Mollusca and human RXRs. RXR1 group contains only parasitic Platyhelminth RXRs, which suggests that parasitic Platyhelminth RXR2 duplicated after the split of their common ancestor with free-living Platyhelminths. In free-liv-  3.7.7. 2DBD-NR. Two 2DBD-NRs were identified in Rhabditophora, M. lignano and four were identified in S. mediterranea; three 2DBD-NRs were found in parasitic Platyhelminths including Monogenea, Cestoda and Trematoda, respectively. A phylogenetic tree of 2DBD-NRs was constructed with the amino acid sequence of the second DBD, because the second DBD is more conserved than the first DBD. Phylogenetic analysis shows that parasitic Platyhelminth 2DBD-NRs clustered in three groups: 2DBDa (Schistosoma mansoni 2DBDα orthologues), 2DBDb (S. mansoni 2DBDβ orthologues) and 2DBDg (S. mansoni 2DBDγ orthologues) groups. Parasitic Platyhelminth 2DBD-NRa and 2DBD-NRb groups contain members of three classes of parasitic flatworms, but 2DBD-NRg group contains 2DBD-NRs of all the four classes of Platyhelminths and members of the Mollusca. This result suggests that Parasitic Platyhelminth 2DBD-NRg is an ancient gene, and 2DBD-NRa and 2DBD-NRb were formed by a second round of duplication. In free living Platyhelminths, both Rhabditophora M. lignano 2DBD-NRs are clustered in 2DBD-NRg group, but the four S. mediterranea 2DBD-NRs are clustered with different parasitic helminth 2DBD-NR groups. This result suggests that M. lignano 2DBD-NR gene underwent duplication after a split of S. mediterranea and parasitic Platyhelminths. For the four S. mediterranea 2DBD-NRs, one is clustered in parasitic Platyhelminth 2DBD-NRg group, one is clustered in 2DBD-NRb group and two are clustered with 2DBD-NRa group. Since the two S. mediterranea 2DBD-NRs (2DBD-NRa1 and 2DBD-NRa2) in 2DBDa group form a polytomy, it suggests that 2DBD-NRa underwent another round of duplication and formed two 2DBD-NRs as a common ancestor of S. mediterranea and parasitic Platyhelminths and then one 2DBD-NRa was lost in a common ancestor of parasitic Platyhelminths (S26 Fig). The DBD sequence length is different among the members of different 2DBD-NR groups, this further supports the phylogenetic analysis. The phylogenetic analysis of 2DBD-NRs in this study is consistent with the analysis of Platyhelminths and Mollusca 2DBD-NRs [67,68].
A scheme represents the phylogeny of Platyhelminths. The twenty-four ancient NR genes in a common ancestor of Platyhelminths are indicated in square box on the left of the figure. NR gene gained by gene duplication is shown above the branch of different flatworm lineages and the gene lost is shown under the branches (italic and strikethrough). The number behind flatworm species/families in parentheses indicates the number of NRs identified.

Discussion
In this study, we identified NRs in different species of Platyhelminths and performed sequence and phylogenetic analysis of them. Phylogenetic analysis of the DBD of NRs using DBD sequences was carried out with Bayesian inference and Maximum Likelihood method, Comparison of the two methods, Bayesian inference highly support phylogenetic analysis of NRs using only DBD sequences. (S3 File). This study shows that NRs in Platyhelminths have orthologues in Deuterostomes, arthropods or both, and the NRs in Platyhelminths diverged into two different evolutionary lineages: 1) Gene duplication and lost; 2) NR gene amplification and divergence.
NRs in Rhabditophora S. mediterranea and parasitic Platyhelminths follow the first evolutionary lineage: gene duplication and lost, the events occurred in different flatworm lineages. For example, fax-1 was lost in a common ancestor of S. mediterranea and parasitic Platyhelminths, NHR236 was lost in Cestoda and Trematoda, and DSF was lost in Cestoda. In parasitic Platyhelminths, extensive NR gene loss occurred in Cestoda Hymenolepididae and Taeniidae families, e. g. RXR, Coup-TF and FTZ-F1 were retained in most species of Platyhelminths, but they were missing in the cestode families Hymenolepididae and Taeniidae.
Comparison of NR complement in different Platyhelminth families shows that NR complement is most conserved in Trematoda. All species of Trematoda share the same NR complement except that a HR96 member was lost in Strigeidida (Schistosoma and Trichobilharzia).  [29,69]. DHR96 (NR1J) plays a role in the response to xenobiotics. The regulation of lipid metabolism and xenobiotics is an ancestral function of members in NR1J group, HR96 links environmental conditions to physiology and development [70]. MlHR96 gene amplification in M. lignano may result from the adaption to its environment. NRs in subfamily 3 and 6 were considered lost in Platyhelminths. In this study, we identified four ERRs and a NR6A in Rhabditophora M. lignano. This is the first known occurrence of NRs in subfamily 3 and 6 in Platyhelminths, but it is still not clear whether NRs from subfamily 3 and 6 are present in other Rhabditophora since the genome data is unavailable. This study also shows that divergent NRs are present in different flatworm lineages suggesting that novel NRs were acquired in different flatworms to adapt to the different living environments.
E75/Rev, DHR3/ROR and EcR/LXR, which are present in Deuterostomia, Arthropods and Lophotrochozoa Mollusca, are missing in Platyhelminths. In insects, E75, HR3 and EcR are directly involved in the control of the ecdysone pathway. E75 acts as a repressor of DHR3 and may through direct interaction [70]. RAR, PPAR, ER, which are missing in Arthropods but retained in Lophotrochozoa Mollusca, are also missing in Platyhelminths. In vertebrates, RARs are known to bind retinoic acid (RA). RA is a morphogen derived from vitamin A, it controls the patterning of the anteroposterior axis and the differentiation of various cell types [71]. Study of Mollusca Nucella lapillus RAR (NlRAR) showed that NlRAR binds to NR response elements as a heterodimer with RXR, but it does not bind all-trans retinoic acid or other retinoids [71]. In vertebrates, PPARs form a heterodimer with RXR binding their response elements, and their ligand includes free fatty acids, eicosanoids and Vitamin B3. In Mollusca N. lapillus, PPAR-responsive pathways is related to tributyltin (TBT) induced imposex [72]. Vertebrate ERs are activated by estradiol (E2) and have important roles in development of the nervous system and secondary sexual traits, but Mollusca ERs cannot bind estrogen, they are constitutively active and retained the ability to regulate their own gene transcription [73].
ERRs are orphan receptors in Chordata and Ecdysozoa, they play important roles in regulation of neurogenesis and metabolism in Chordata and are involved in control of larval growth in Ecdysozoa. The function of Lophotrochozoa ERRs is unknown [73]. In this study, four ERRs are identified in Platyhelminths (MlERRs), phylogenetic analysis shows that these members are closely related ERRs. The further characterization will reveal their role in development of Platyhelminths.
The NR2E6 gene was first identified in the genome of the honeybee Apis mellifera and then in other insects, but it was missing in Drosophila. The missing of NR2E6 in the major model organisms delayed the identification and functional analysis of this protein. Sometimes NR2E6 is named as PNR-like or PNR, but the true insect homolog of vertebrate PNR is HR51 (NR2E3) [70]. We demonstrated that a novel NR2E member (NHR236, orthologue of nematode Caenorhabditis elegans HR236) was present in Cnidaria, Arthropoda, Platyhelminths, Mollusca and Echinodermata [14]. Insect NR2E6 is an orthologue of NHR236 and all of NHR236 orthologues possess an unique P-box sequence (CDGCRG) in the DBD region.
Previously, we isolated a partial cDNA of S. mansoni HR96b (SmNR96b) [12], this member has a CHC2 zinc finger motif in the second zinc finger of DBD. In this study, we show that all parasitic Platyhelminth SmHR96b orthologues contain this novel motif. Whether the function of this new type of motif in NRs may change the DNA binding properties awaits further study. . We identified the same sequences but our phylogenetical analysis showed that they belonged to NR1E (E78) group. (4). Cheng, Y., et al. identified 20 NR1Js (HR96s) in M. lignano including one sequence without a DBD sequence and 5 members each with an atypical P-box sequence which is different from the typical HR96 P-box sequence. We identified 16 HR96s in M. lignano, all of them possessed a typical HR96 P-box sequence. We also identified the same 5 members with an atypical P-box sequence that Cheng, Y., et al. identified, but put them into a divergent NR group. (5). Cheng, Y., et al. identified 10 NR3 members in M. lignano including four members that each possessed a typical ERR P-box sequences and 6 members that each possessed an atypical P-box sequence. We identified the same members but put the six members that possessed an atypical P-box sequence into a divergent NR group. (6). Cheng, Y., et al. identified a NR8 member, we identified the same member but our phylogenetical analysis showed it was a divergent member. (7). Cheng, Y., et al. identified 2 NR0 members. We identified the same members but our phylogenetic analysis showed one of them belong to subfamily 6 and the other one is a divergent member because it possessed an atypical P-box sequence.