* Department of Neuroscience, Unit of Pharmacology
Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, Uppsala, Sweden
Correspondence: E-mail: dan.larhammar{at}neuro.uu.se.
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Abstract |
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Key Words: neuropeptide Y G proteincoupled receptor 2R hypothesis linearized tree gnathostome chondrichthyes Squalus acanthias
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Introduction |
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Despite the evidence, the 2R hypothesis remains controversial (Hughes 1999; Hughes, da Silva, and Friedman 2001; Martin 2001; Pennisi 2001). Alternative hypotheses include independent gene duplications followed by parallel aggregation into similar clusters (Hughes 2000) or independent chromosome or block duplications rather than simultaneously in tetraploidizations (Skrabanec and Wolfe 1998; Smith, Knight, and Hurst 1999). The arguments are discussed in recent reviews (Wolfe 2001; Larhammar, Lundin, and Hallböök 2002).
Another issue is when the duplications took place. Several authors have suggested that they occurred before the origin of gnathostomes but after amphioxus branched off (Holland et al. 1994; Sidow 1996; Spring 1997). This is supported by the presence of a single Hox cluster in amphioxus as opposed to four in tetrapods (Garcia-Fernàndez and Holland 1994). This is also true for the large chromosomal region carrying the major histocompatibility complex (Abi-Rached et al. 2002). After the divergence of amphioxus and before the appearance of gnathostomes, the agnathan (jawless fishes) hagfishes and lampreys appeared, either independently or as a monophyletic group (Delarbre et al. 2002). It has been proposed that the first tetraploidization took place before the agnathans diverged and that the second tetraploidization happened subsequently in the lineage leading to gnatho-stomes (Holland et al. 1994). This interpretation is complicated by the discovery of three to four Hox clusters in lampreys, but these clusters may have occurred independently (Force, Amores, and Postlethwait 2002; Irvine et al. 2002; Sharman and Holland 1998).
The time point for the second proposed tetraploidization is also uncertain, as only two Hox clusters have yet been identified in any cartilaginous fish (Kim et al. 2000). A recent review placed the second tetraploidization after divergence of sharks (Pennisi 2001). Regardless, cartilaginous fishes and agnathans are important groups for determining time points and mechanisms for the increase in vertebrate gene number.
We have previously investigated the evolution of the neuropeptide Y (NPY) family of neuroendocrine peptides in vertebrates and found that the first gene duplication leading to NPY and peptide YY (PYY) probably took place before the origin of lampreys, as both the river lamprey and gnathostomes have NPY and PYY (Cerdá-Reverter and Larhammar 2000; Larhammar 1996; Söderberg et al. 1994). More recently, we have described an NPY receptor in the river lamprey Lampetra fluviatilis that appears to be a pro-ortholog of the tetrapod receptor Y4 and the teleost receptor Yb (Salaneck et al. 2001). Both NPY peptide and receptor genes belong to paralogons that have likely arisen by duplications in early vertebrate evolution, as the genes for NPY and PYY are linked to Hox clusters (Larhammar 1996), and the receptor genes belong to the paralogon consisting of human chromosomes Hsa4, Hsa5, Hsa8, and Hsa10 (Wraith et al. 2000).
The NPY receptors belong to the superfamily of G proteincoupled receptors that include three Y1 subfamily subtypes named Y1, Y4, and Y6 and are located on human chromosomes 4, 10, and 5, respectively (Wraith et al. 2000). These three mammalian receptors are approximately 50% identical to each other as well as to three teleost fish receptors Ya, Yb, and Yc (Lundell et al. 1997; Ringvall, Berglund, and Larhammar 1997; Starbäck et al. 1999). Relationships between the mammalian and teleostean receptors have been unclear. Ya may be an ortholog of mammalian Y4, whereas Yb and Yc are closely related to each other, and their teleost pro-ortholog, Yb/c (represented in our trees by cod Yb/c), could be the fourth member of the quartet comprising this paralogon. The mammalian Yb/c ortholog was probably lost. The receptors named Y2 and Y5 are more distantly related to the Y1 subfamily, and Y3 has only been defined pharmacologically and probably does not exist as a separate gene (Larhammar et al. 2001).
As the phylogenetic resolution of agnathan sequences relative to the vertebrate gene duplication events is often ambiguous in gene family trees (Ono-Koyanagi et al. 2000; Germot et al. 2001; Neidert et al. 2001; Salaneck et al. 2001), it is important to obtain additional sequence information from agnathans and cartilaginous fishes. The latter group is poorly represented in molecular evolutionary studies. We describe here the cloning and phylogenetic analyses of three NPY receptor subtypes belonging to the Y1 subfamily in the spiny dogfish, Squalus acanthias. These were identified as orthologs of mammalian receptors Y1, Y4, and Y6, thereby showing that block duplications of this paralogon preceded the origin of gnathostomes. We also describe unexpected anatomical distribution of receptor mRNA.
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Materials and Methods |
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Sequence Alignments
We made separate alignments of the Y1 subfamily (Y1, Y4, and Y6, hence "146") and all available vertebrate NPY receptors (adding Y2 and Y5, hence "12456") to assess alignment uncertainty and to reduce the effect of this uncertainty on analyses requiring only the smaller data set.
The alignments were made with ClustalW (Thompson, Higgins, and Gibson 1994) and optimized with MultiClustal (Yuan et al. 1999). The MultiClustal alignments and parameters were similar to those optimized by hand with ClustalW alignment quality guides. The parameter values and models used in the creation of four alignments are shown in table 1. Because gaps were contained almost exclusively in loops and at the termini, all gap-free alignments consisted almost entirely of transmembrane regions and were highly consistent with one another.
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The MultiClustal-optimized alignments of the data sets without DreYa ("" and "
") were different both qualitatively and quantitatively from those of the data sets with DreYa (table 1). For analyses without DreYa, we therefore used these separate alignments.
We initially removed all gap-containing sites. This forced us to exclude two truncated or incomplete sequences (human Y6 and sturgeon Y1) from all data sets. We later found it necessary to retain gaps in the analysis of the largest data set, 12456DreYa (see below). In analysis of gap-containing alignments, we ensured that all software we used was consistent in treating gaps as missing data.
Phylogenetics
Likelihood Analysis
Using Tree-Puzzle to optimize maximum-likelihood (ML) branch lengths with initial Neighbor-Joining (NJ) tree of the 12456DreYa data set computed with NEIGHBOR from JTT-F ML pairwise distances computed in Tree-Puzzle, we found that the JTT-F model (Jones, Taylor, and Thornton 1992) of protein evolution had a much larger likelihood with the data (JTT-F: log
) than other available models available in Tree-Puzzle (WAG-F: log
; VM-F: log
; and BLOSUM-F: log
). This result did not change when these other models were used to calculate the initial pairwise distances.
We conducted likelihood ratio tests for site-rate heterogeneity using initial likelihood trees estimated in PROTML version 2.3b3 (Adachi and Hasegawa 1995), using local rearrangement search and default parameters, from initial Neighbor-Joining trees estimated with NJDIST version 1.2.5 (Saitou and Nei 1987). Both initial pairwise distances and likelihood trees were estimated with PROTML using the JTT-F model. The resulting PROTML-estimated tree topologies were then input to Tree-Puzzle for likelihood estimation trees with and without a discrete approximation (exact estimation, eight intervals) to a gamma distribution of site-rate heterogeneity.
Assuming the likelihood ratio test (LRT) statistic for JTT-F versus JTT-F- is distributed according to a 50:50 mixture of the
20 and
21 distributions (denoted
21 as in Goldman and Whelan [2000]), then we could reject the null hypothesis of site-rate homogeneity in all three data sets with very high confidence. The LRT statistics and initial ML estimates (and their standard errors) of the alpha shape parameters for the three data sets are presented in table 2, wherein P(
) = [P(
)/2] (Goldman and Whelan 2000). Similar results were obtained with the gapped 12456 alignment (calculated from an NJ tree from PAUP*).
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Bayesian Inference of Phylogeny
We analyzed the full (gap-containing) 12456DreYa alignment in MrBayes version 3.0B (Huelsenbeck and Ronquist 2001) with the JTT-F-
model and the gamma shape parameter
coestimated with the trees. The program ran for 117,140 steps with a tree sampled every 20 generations. The first 857 trees ("burn-in") were discarded as a generous estimate of the onset of stationarity, leaving 5,000 trees for analysis. The program was initialized from a random tree. As a comparison and control against local maxima, short runs were initialized from a "user tree," which was an initial PROTML-optimized topology made as described above for likelihood ratio testing, and from an additional random tree.
Distance and Parsimony Analysis
Consensus NJ trees were estimated from 100 bootstrap replicates generated from the data sets in SEQBOOT from the PHYLIP package (Felsenstein 1993), using ML distances under the JTT-F- model with initial alpha parameter estimates as given above or in table 2. Pairwise distance matrices were calculated with Tree-Puzzle running under PUZZLEBOOT. NJ trees were calculated with random addition of sequences using NEIGHBOR and a consensus tree generated from them under extended majority-rule with CONSENSE, both from PHYLIP. Bootstrapped consensus parsimony trees (100 replicates) were obtained using PAUP* (Swofford 2000) with mean character values, "ACCTRAN" or "accelerated transformation," and random addition of sequences.
Phylogenetic Tests of the Molecular Clock and Linearized Trees
We estimated absolute and relative times of divergence of the Yb,Yc clade and the Y4 clades and of the Y1,Y6 clade and the Y4,Yb,Yc clade by generating a linearized tree (Takezaki, Rzhetsky, and Nei 1995) with the Y2 and Y5 sequences as outgroups and calibration dates from estimated Squalus and Lampetra fossil divergence times.
The topology of the Bayesian consensus tree of the 12456DreYa data set (gapped) and its corresponding data set were automatically converted and reordered to the MEGA-style format required by the LINTREE package using custom Perl software (FAS2LTRE, NEWICK2MEGA, and LTRECONFORM) available from David H. Ardell. Two-cluster and branch-length tests were computed with TPCV and BRANCH from the LINTREE package. We used the amino-gamma distance with the alpha estimate from gap-free data (table 2) because LINTREE eliminates gap-containing sites.
Three rounds of sequence elimination were necessary to produce a nearly ultrametric linearized tree. In the first round, the two-cluster test indicated heterogeneity between the slow-evolving Squalus Y6 and the more quickly evolving mammalian Y6 sequences (P < 0.01). Peccary y6, in this group, was also the only significantly fast (or slow) evolving sequence compared with the branch average and so was eliminated using COLLAPSE (available from David H. Ardell). In the second round (reusing the same alpha estimate), no individual node or branch was significantly heterogeneous at the 1% level, and overall rate heterogeneity as quantified by the U statistic for the two-cluster test was not significant (;
) but the U statistic for the branch-length test was still significant (
;
), with the Squalus Y4 sequence evolving quickest and the Cod Yb sequence evolving slowest. Cod Yb was most atypical overall and was removed for a third round. In the third and fourth rounds, heterogeneity between the Squalus and mammalian y/Y6 sequences and/or overall rate heterogeneity were still significant at the 5% level, requiring elimination of the mouse y6 and then Squalus Y6 in the third and fourth rounds, respectively. In the fourth round, no clusters or branches exhibited rate heterogeneity at the 5% level, nor did the data set overall, by either test.
RT-PCR
One female Squalus acanthias was captured in the Skagerack Sea (Larssons Fisk HB, Mollösund, Sweden). Tissue samples were placed in RNAlater (Ambion). Total RNA preparations were made with ca 50 mg from each tissue according to RNeasy kit protocol (Qiagen) including DNase treatment to eliminate genomic DNA contamination. Primers designed from the deduced Squalus sequences were used (primer sequences available upon request) in Titan One-tube RT-PCR reactions (Roche Biochemicals) with ca 20 pg total RNA in each reaction according to protocol. Specific primers for Squalus acanthias myelin basic protein (MBP) was used as a positive control (Spivack et al. 1993) (sequences upon request). For a negative control, all utilized RT-PCR primer combinations were tested in standard PCR conditions with Taq polymerase (Gibco BRL) on the RNA preparations. Also, as a second negative control, all RT-PCR reactions were run without the preliminary reverse transcriptase step. The products were analyzed on standard 1.5% agarose gels and blotted overnight to nylon filters. Filters were hybridized with 32P-labeled probes at 65°C in ExpressHyb buffer overnight and exposed to film (Amersham) overnight at -70°C.
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Results |
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Monophyly and Rooting of the 146 Clade
Figure 2 shows a cladogram of the complete NPY data set without Danio rerio Ya ( gapped). Support for monophyly of a Y1,Y6,Y4 clade ("146" or "Y1 subfamily") was unequivocal.
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The 146 clade could not be rooted reliably with the gap-free version of this alignment. In the gap-free likelihood tree made with PROTML, the root fell between the Lampetra Y receptor sequence and the other Y4,Yb,Yc sequences, with no support for the placement as determined by RELL relative likelihood values (data not shown). The NJ tree gave a result consistent with that shown in figure 2, but the support for placing the root outside the Lampetra Y receptor sequence was only slight to nonexistent, at 64. The parsimony results, on the other hand, were less affected by the presence or absence of gaps than those of the other methods. Also, in the gap-free analysis, the monophyly of the 146 clade remained unequivocally supported by all methods of analysis. In the Bayesian analysis of this data set, the posterior mean log-likelihood of sampled trees was ln L = -13798 with a 95% credible interval of ln L (-13812; -13785). The estimated posterior mean shape parameter for the gamma distribution of site-rates was
= 0.89 with a 95% credible interval of
(0.75; 1.03).
Phylogenetic Placement of Squalus acanthias Sequences
Unrooted likelihood phylograms of the 146 clade are shown in figure 3. With most branches, particularly those of the Squalus acanthias and Lampetra fluviatilis sequences, all methods gave consistent and reliable results. The three SacY1 subfamily homologs segregate clearly and consistently near the roots of the Y1, Y4 + Ya, and Y6 clades.
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Absolute and Relative Divergence Time and Rate Estimates Within the Y1-Subfamily
As described in the methods section, the Y6 clade was particularly heterogeneous in evolutionary rate, consistent with pharmacological data suggesting partial or complete inactivity of this subtype in mammals. The Yb,Yc sequences evolved significantly slower than did Y4 sequences (), by the two-cluster test.
Table 3 shows estimated duplication dates within the Y1 family of NPY receptors along with statistical results. The estimated dates are from the linearized Bayesian consensus tree (fig. 4) of the 12456DreYa data set with the most heterogeneously evolving sequences removed as described in Materials and Methods. Dates for the Y4/Yb,Yc split were calculated using h and the paleontological dates for the chondrichthyan-osteichthyan split (385 MYA) and the Lampetra-gnathostome split (450 MYA).
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The estimated relative heights and standard errors of the Y1/Y6 split and the Y4/Yb,Yc splits are 0.431 ± 0.033 and 0.431 ± 0.044, respectively. Thus, assuming the placement of the lamprey sequence is correct, we could not reject simultaneous divergence of the two clade pairs.
This analysis is sensitive to the placement of the Lampetra Y sequence. Under the assumption of monophyly of this sequence with the Y1,Y6 clade (generated by a consensus of the Bayesian credible trees constrained by this monophyly assumption in PAUP*) a similar dating analysis rejected simultaneity of duplications creating the Y1,Y6 clade and Y4,Yb,Yc clade.
Reverse Transcriptase PCR
RT-PCR revealed widespread mRNA expression for the three isolated genes (fig. 5). RT-PCR for Sac MBP was used as a positive control for brain tissue (not shown). The Y1 gene showed expression primarily in liver and kidney/interrenals (the piscine adrenal homolog), but not in the CNS. SacY4 was expressed in brain, retina, liver, and skeletal muscle. The SacY6 gene showed expression in retina and gastrointestinal tract and to a lesser extent in the kidney and interrenal tissue. Blotting to nylon filters and subsequent hybridization confirmed the identity of the bands seen on the agarose gels with 32P-labeled probes for the respective genes. The signals detected on the autoradiograms confirmed the patterns seen on the gels (fig. 5).
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Discussion |
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Many other regions also exist in four copies in mammals (Lundin 1993; Popovici et al. 2001a, 2001b). At least six such quartets, or paralogons, have been identified (Popovici et al. 2001a, 2001b). A parsimonious explanation would be duplications of the entire early vertebrate genome via two tetraploidizations, followed by extensive gene loss and chromosomal rearrangements (Ohno 1970; Holland et al. 1994; Sidow 1996; Pebusque et al. 1998; Abi-Rached et al. 2002).
The paralogon harboring the Y receptors consists of four related chromosome regions, but a fourth Y1 subfamily receptor has not yet been identified in mammals, chicken, or spiny dogfish. It is possible that one of the three teleost Y1-like receptors Ya, Yb, and Yc (Lundell et al. 1997; Ringvall, Berglund, and Larhammar 1997; Arvidsson et al. 1998; Starbäck et al. 1999) could correspond to the fourth member of this paralogon (fig. 6). However, the teleost receptors have previously been difficult to classify relative to the mammalian subtypes. We therefore investigated whether their phylogenetic positions could be better resolved by including the three spiny dogfish receptors and our recently reported Lampetra fluviatilis Y receptor (Salaneck et al. 2001), proposed to be a Y4,Ya,Yb,Yc pro-ortholog.
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The position of the Gmo Yb sequence from the cod (Arvidsson et al. 1998) suggests that the duplication resulting in Yb and Yc in zebrafish occurred after the split between these two teleosts and not as a result of the basal teleost tetraploidization (Amores et al. 1998; Taylor et al. 2001). However, data from more teleosts are needed before the time point for the Yb/Yc duplication can be determined with certainty. Whether additional duplicates of any of these four Y1 subfamily genes exist in teleosts, as a result of their additional tetraploidization (fig. 6), remains to be investigated. According to the proposed duplication scheme, the Yb gene should have arisen before the divergence of Chondrichthyes. Further studies are needed to see if Yb still exists in any species of this class or whether it has been lost, as seems to be the case in mammals. The chromosomal locations of the zebrafish Ya, Yb, and Yc genes (Starbäck et al. 1999) do not at present clarify the relationships to their mammalian genes.
The phylogenetic position of the Lampetra fluviatilis receptor was not clearly resolved by our data. However, the Bayesian analysis provides clear support of its membership in the Y4,Ya,Yb,Yc clade at a posterior probability of 0.94. If the lamprey sequence is indeed a Y4 receptor, the first chromosome duplication (tetraploidization) would have taken place before the lampreys diverged from the gnathostome ancestor and the second chromosome duplication (tetraploidization) took place in the gnathostome lineage (fig. 6). However, the idea that one tetraploidization took place on each side of the lamprey branch point is questioned by the finding of three to four Hox clusters in the sea lamprey, Petromyzon marinus (Force, Amores, and Postlethwait 2002; Irvine et al. 2002). We should be able to shed more light on this issue by cloning more Y1-like lamprey sequences. In any event, it seems clear that both chromosome duplications took place before the radiation of gnathostomes (fig. 6).
To determine whether the four clades of the Y1 subfamily arose via duplication of two pro-orthologs, a linearized tree was generated. Using this tree (fig. 4), we found that the Y1, Yb + c, Y4, and Y6 clades arose simultaneously (i.e., we could not falsify the hypothesis that the two deep branches became four at a single time point). The so-called 2 + 2 topology we found, also denoted (AB)(CD), has not been consistently obtained for other gene families and quadrupled chromosomes (Hughes, da Silva, and Friedman 2001), although variable evolutionary rates may also explain this (Abi-Rached et al. 2002). This result depends on the monophyly of the lamprey sequence with the Y4,Yb,Yc clade; we were able to reject simultaneity on the hypothesis that the lamprey sequence groups with the Y1 and Y6 receptors.
The tissue distribution of the three shark receptor mRNAs, as determined by RT-PCR, revealed unexpected differences from mammals and chicken. Mammalian Y1 is primarily expressed in the CNS (Mikkelsen and Larsen 1992). We could not detect SacY1 expression in the brain, but instead in peripheral tissues (fig. 5). This was unexpected, considering that receptor binding studies performed on CNS sections of the smooth dogfish Mustela canis seemed to identify Y1-like binding in the hypothalamus (McVey et al. 1996).
Y4 expression in the shark was also apparently different than that in mammals, where it is found predominantly in the gastrointestinal tract (Feletou et al. 1999). We detected SacY4 in retina, muscle, renal/interrenal tissue, and the brain. Mammalian Y4 is a receptor for the gastrointestinal peptide PP (Lundell et al. 1995), which has been observed only in tetrapods (Larhammar 1996). We hypothesize that PP hijacked the preexisting Y4 receptor at some point during tetrapod evolution, concomitant with divergence in function of the Y4 receptor. This is consistent with the different expression pattern of the shark Y4 receptor, the first nontetrapod Y4 to be cloned.
SacY6 expression was detected in retina, GI tract, and kidney/interrenal tissues. Comparisons with mammals are uncertain since, in most mammals, Y6 is either a pseudogene or has not been allocated a physiological correlate (Matsumoto et al. 1996; Rose et al. 1997; Starbäck et al. 2000; Wraith et al. 2000). The apparent reduced importance of mammalian Y6 is further supported by the fact that all known Y6 pseudogenes have become nonfunctional by distinct mutations (Larhammar et al. 2001).
An explanation for these divergent expression patterns, considering that the duplications occurred shortly before the gnathostome radiation, could be that subfunctionalization of the Y1 subfamily genes or their acquisition of novel functions had probably not yet occurred by the time of the Chondrichthyan split from all other gnatho-stomes (Osteichthyes, i.e., Sarcopterygians plus Actinopterygians).
In summary, we have isolated elasmobranch orthologs to all three Y1 subfamily receptor genes previously found in tetrapods. Orthology is strongly supported by phylogenetic analyses, overall sequence identity as well as rare genomic changes in the sequences, such as an intron and deletions. The localization of these genes on related chromosome segments in mammals and chicken, along with their presence in the spiny dogfish, suggest that the gene family expanded by en bloc duplications early in vertebrate evolution. Together with data from other gene families, this supports the theory of the vertebrate genome having expanded by means of chromosomal or genomic duplications before the rise of gnathostomes.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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