General and Comparative Endocrinology 148 (2006) 306–314

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Minireview

Are lungWsh living fossils? Observation on the evolution of the opioid/orphanin gene family

Jenny Lee a, Jasem Alrubaian b, Robert M. Dores c,¤

a Division of Cardiology, University of Colorado Health Sciences Center, Denver, CO 80262, USAb Department of Biological Sciences, University of Kuwait, Kuwait City, 13060, Kuwait

c Department of Biological Sciences, University of Denver, Denver, CO 80210, USA

Received 8 June 2006; revised 16 July 2006; accepted 19 July 2006

Abstract

This minireview considers the possibility that there is a correlation between the slow rate of morphological change and speciationevents that has been occurred within the lungWsh lineage since the Permian period, and the apparent slow rate of divergence in the aminoacid sequences of lungWsh opioid precursor sequences. The status of lungWsh as “living fossils” is considered.© 2006 Elsevier Inc. All rights reserved.

Keywords: Evolution; Proenkephalin; Prodynorphin; Proopiomelanocortin; LungWsh; Amphibian; Mammals

1. What is a “living fossil?”

The challenges associated with reconstructing the evolu-tion of a gene family are compounded by the fact that geneswithin a family may be evolving at diVerent rates, and, moreimportantly, the primary source for conducting these analy-ses are gene sequences obtained from contemporary speciesthat most certainly are not identical to their ancestral prede-cessors. Although some insights on gene evolution havebeen made from the analysis of DNA from fossilized spe-cies, the integrity of “ancient DNA” that is in the tens tohundreds of millions of years in age is not adequate to iden-tify the sequences of ancestral genes during the early radia-tion of the various lineages of the chordates, for example.However, when a gene resurrection algorithm approach(Thornton, 2004) is used to evaluate gene sequence dat-abases compiled from extant vertebrate species, it is possibleto make predictions on the likely sequence of ancestralgenes. This approach has been used with considerable suc-cess to reconstruct the evolution of glucocorticoid and min-

* Corresponding author. Fax: +1 303 871 3471.E-mail address: rdores@du.edu (R.M. Dores).

0016-6480/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2006.07.010

eralocorticoid hormone receptors in chordates (Bridgehamet al., 2006). For this type of analysis, the identiWcation ofextant species with long fossil records that appear to beevolving slowly would be of considerable utility.

Darwin (1859) introduced the concept of a “living fossil”as an extant species that belongs to an old lineage that hasundergone slow rates of evolution in terms of the diversiW-cation of the lineage into new families and genera. Asreviewed by Stanley (1998), the debate on the nature of liv-ing fossils has largely focused on comparisons of the ratesof morphological change and the degree of speciation asrecorded in the fossil record of the lineage. For example,Simpson observed a correlation between the rate of mor-phological change in lungWsh over the past 400 millionyears and the degree of diversiWcation of Infraclass Dipnoiin terms of the number of genera that have evolved overthis period (Simpson, 1944, 1953). Based on this analysis,the highest degree of morphological diversity observed forthe lungWshes occurred in the middle to late Devonian. Bycontrast, from the Permian Period to the present there hasbeen a remarkable degree of morphological stasis in thebody plan of the lungWshes. During the Devonian this spikein morphological diversity was matched by the highest levelof species diversity for the lungWshes as measure by the

J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314 307

number of genera identiWed in the fossil record (Simpson,1953). By the Permian the number of lungWsh genera haddeclined by over 70%, and at present there are only threegenera of extant lungWsh (Nelson, 1994). If apparent stasisin the phenotype of the genes that regulate morphology hasoccurred in lungWsh, would this apparent stasis be evidentin genes that do not code for morphological features? Totest this possibility a comparison of genes cloned from twoof the extant genera of lungWsh, Neoceratodus and Prot-opterus, was conducted. The opioid/orphanin gene familywas selected for this analysis.

2. The opioid/orphanin gene family

The opioid/orphanin gene family is a collection ofnuclear genes that encode the sequences of opioid-relatedneuropeptides; ligands that bind to opioid receptors in thecentral nervous system and at peripheral target tissues(Dores et al., 2002; Waldhoer et al., 2004). In mammalsthere are four genes in the family: proenkephalin, prody-norphin, proorphanin, and proopiomelanocortin (POMC).In each gene, the opioid sequence(s) is embedded in a pre-cursor protein that has a signature set of cysteine residueslocated in the N-terminal region (Douglass et al., 1984).The proenkephalin gene encodes the pentapeptide opioidsmet-enkephalin (YGGFM) and leu-enkephalin (YGGFL),and two C-terminally extended forms of met-enkephalin(Met-enk-7 and Met-enk-8; Noda et al., 1982). The prody-norphin gene encodes three C-terminally extended forms ofleu-enkephain: �-neoendorphin, dynorphin A and dynor-phin B (Kakidani et al., 1982). In mammals, the proorpha-nin gene encodes a seventeen amino acid polypeptide,orphanin, that has the N-terminal sequence FGGF (Meu-nier et al., 1995; Mollereau et al., 1996). Mammalianorphanin does not bind to opioid receptors, but does bindto the ORL receptor, a member of the opioid receptor genefamily (Reinschied et al., 1995). The POMC gene encodesthe opioid �-endorphin, a C-terminally extended form ofmet-enkephalin, and the melanocortin ligands (ACTH,�-MSH, �-MSH, �-MSH; Nakanishi et al., 1979). In addi-tion to mammals, the deduced amino acid sequences forproenkephalin, prodynorphin, and POMC have been deter-mine from cDNAs cloned from the brain and pituitary ofseveral vertebrate groups including the lungWsh (for reviewssee: Dores and Lecaude, 2005; Khalap et al., 2005).

3. The phylogeny of the lungWsh

LungWsh emerged in the Devonian, a period marked bythe emergence of two major types of bony Wsh; the lobe-Wnned Wsh (Sarcopterygii) and the ray-Wnned Wsh (Actin-opterygii). LungWsh were derived from a lobe-Wnned Wshancestor at least 400 million years ago (Carroll, 1988) andhave a continuous and plentiful fossil record to the present.Extant species belong to Infraclass Dipnoi, and are segre-gated into two orders: Ceratodontiformes and Lepidosiren-iforms. There is general consensus that these orders had a

monophyletic origin and have been separated for at least120 million years (Carroll, 1988). Order Ceradontiformeshas one extant species, the Australian lungWsh Neocerato-dus forsteri. Order Lepidosireniforms is represented by Wveextant species in two genera; the South American lungWsh,Lepidosiren paradoxa, and four African lungWsh species inthe genus Protopterus. The species Protopterus annectes wasused for this analysis. Extant lungWshes live in freshwaterhabitats, hence the breakup of the super continent Pangaealed to the geographic isolation of the Australian lungWshspecies from the South American and African speciesbetween 100 and 120 million years ago. The later time pointwill be used for this analysis.

4. Are lungWsh living fossils?

For this analysis, the deduced amino acid sequences forproenkephalin, prodynorphin, and POMC will be com-pared in two species from the following groups: Dipnoi(Neoceratodus forsteri and Protopterus annectes); anuranamphibians (Xenopus laevis and Bombina orientalis); andplacental mammals (Rattus norvegicus and Homo sapiens).As presented in Fig. 1, the lungWshes and the tetrapods(amphibians, reptiles, birds, and mammals) are a monophy-tic assemblage that was derived from the ancestral Sarco-pterygii. The lungWshes can be found in the fossil record atleast 400 million years ago and have diverged from the lobeWnned Wsh ancestors that would give rise to the ancestraltetrapods (Brinkman et al., 2004). The radiation of theancestral tetrapods into the extant groups of amphibians iscomplex and is represented by the dashed lines. There ishowever general agreement that the modern amphibians(Orders Anura, Caudata, Gymnophiona) are a monophy-letic assemblage (Duellman and Truab, 1994), and withinthis group the anuran genera, Pipidae (represented byXenopus laevis) and Discoglossidae (represented by Bom-bina orientalis), appear in the fossil records by 140 millionyears ago. The ancestral tetrapods also gave rise to theancestral amniotes at some point in the CarboniferousPeriod. The radiation of the ancestral reptiles into the mod-ern reptile, bird, and mammal groups is also rather complex(dashed line). However, it would be reasonable to assumethat the various orders of placental mammals are a mono-phyletic group, and that the rat and human lineages lastshared a common ancestor around 85 million years ago(Springer et al., 2003; Kemp, 2005). If all members of thegene family are evolving at the same rate, then the rate ofchange as expressed as mutations per millions of years forthese paralogs should be uniform within a taxonomicgroup. If orthologs within the opioid gene family are evolv-ing at the same rate, then the rate of accumulation of muta-tions in each ortholog should be uniform within the threedata sets.

Fig. 2 provides a comparison of the deduced amino acidssequences for proenkephalin (Sollars et al., 2000; Doreset al., 2000), prodynorphin (Dores et al., 2004) and POMC(Dores et al., 1999) in the two species of lungWsh. As

308 J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314

observed for other gnathostomes (Khalap et al., 2005),lungWsh proenkephalin encodes seven opioid sequences:Wve copies of the met-enkephalin sequence and two C-ter-minally extended forms of met-enkephalin. The enkephalinsequences are identical in both species, and the percentageof sequence identity between the two lungWsh proenkepha-lin sequences is 81%. A simple way to evaluate the diver-gence of the two lungWsh proenkephalin sequences is tocalculate the Unit Evolutionary Period (UEP; Dickerson,1971); that is the millions years required to yield a 1%divergence in primary sequence between the lungWsh proen-kephalin sequences. Using 120 million years as the diver-gence time for the two lungWsh genera and noting that inFig. 2 there is a 19% diVerence in the primary sequence ofthe lungWsh proenkephalin sequences, a 1% amino aciddivergence in these sequences is expected to occur every 6.3million years. The UEP value does not say anything aboutthe occurrence of neutral nucleotide changes nor does itcalculate the number of proposed point mutations requiredto occur in a codon to yield a change in the amino acidsequence at any given position in the protein. It does, how-ever, provide a relative indication of the degree of stabilityof a protein sequence as compared to other proteins, in thiscase, within a gene family.

For example, when the prodynorphin sequences for thetwo lungWsh species are compared (Fig. 2), the UEP valuefor this gene is 4.3 million years. The complete sequence ofAustralian lungWsh prodynorphin indicates that Wve opioidsequences are embedded in this precursor (Dores et al.,2004). There are two copies of the pentapeptide opioid, leu-enkephalin, a �-neoendorphin sequence, a dynorphin Asequence, and a dynorphin B sequence. The partialsequence of the African lungWsh prodynorphin sequenceclearly encodes four of the Wve opioid sequences. When the

partial sequence of African lungWsh prodynorphin is com-pared to the corresponding region in Australian lungWshprodynorphin (positions 45–236), there is 72% primarysequence identify between the two sequences. Since dynor-phin B sequences are highly conserved in gnathostomes(Khalap et al., 2005), it is anticipated that the percent pri-mary sequence identity between these two prodynorphinsequences would be higher.

This prediction was supported by the analysis of thelungWsh POMC sequences presented in Fig. 2. Bothsequences follow the sarcopterygian organizational planfor POMC (Dores and Lecaude, 2005) and encode fourmelanocortin sequences (�-MSH, �-MSH, �-MSH, andACTH [�-MSH plus CLIP]), and a �-endorphin sequence.In addition, there is 82% primary sequence identity betweenthe two sequences. Hence, for the lungWsh POMC gene, 6.7million years would be required to produce a 1% change inprimary sequence. Collectively, it appears that the opioidgenes in lungWsh are evolving at similar rates with thecaveat that the rate of divergence of the lungWsh prodynor-phin gene may be an overestimate.

However, the potential relevance of these UEP estimatesin terms of whether the lungWsh opioid genes are evolvingslowly can only be ascertained by calculating these valuesfrom representatives of other taxonomic groups. For exam-ple, the comparison of Xenopus laevis and Bombina orien-talis opioid sequences presented in Fig. 3 indicatesdiVerence rates of evolution for the amphibian opioidgenes. The amphibian proenkephalin sequences follow thesame organization plan as the lungWsh proenkephalinsequences (seven opioid sequences present in the precursor(Martens and Herbert, 1984; Wong et al., 1991; Dores et al.,2001). However, assuming a divergence for the two anurangenera of 140 millions, the UEP value for the amphibian

Fig. 1. Vertebrate phylogeny. The divergence times for the taxa used in this study is presented. The estimates on the divergence times was obtained fromCarroll (1988). Dashed lines indicate that the phylogeny with that is complex and that the taxa being considered are not direct descendents of the ancestralvertebrates presented in this Wgure.

DIPNOI ANURA EUTHERIAN MAMMALSPRESENT RATTUS HOMO

50

100

150MY 200A

250ANCESTRAL AMNIOTES

300

350

400 ANCESTRAL LUNGIFSH

ANCESTRAL SARCOPTERYGIANS

ANCESTRAL TETRAPODS

NEOCERATODUS PROTOPTERUS XENOPUS BOMBINA

J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314 309

proenkephalin sequences would be estimated to be 4.1million years.

The amphibian prodynorphin sequences display manyof the features observed for the lungWsh prodynorphinsequences (Pattee et al., 2003). There are two pentapeptidesequences near the N-terminal region of these precursors.However, in X. laevis there is a novel YGGFI sequence.This opioid sequence is also found in some ray-Wnned Wshprodynorphin sequences (Alrubaian et al., 2006). When thefull length amphibian prodynorphin sequences are com-pared, the level of primary sequence identity is 40% and the

UEP value would be estimated to be 4.1 million years. If thecomparison is limited to positions 29–251 in Fig. 3 in orderto correspond to the same region analyzed in the lungWshprodynorphin sequences, the level of primary sequenceidentity drops to 34%. When this later value is used to cal-culate the UEP for the amphibian prodynorphin sequence,the estimated value is 2.1 million years.

The amphibian POMC sequences (Martens et al., 1986;Kozak et al., 2005) also show the same organization plan asthe lungWsh POMC sequences, and the level of primarysequence identity is 70%, and the estimated UEP for the

Fig. 2. LungWsh opioid precursor sequences. The following lungWsh opioid precursor amino acid sequences were aligned as described in Dores et al. (1996).Australian lungWsh Proenkephalin (AAF44658), African lungWsh Proenkephalin (AAF44657), Australian lungWsh Prodynorphin (AAS18483), AfricanlungWsh Prodynorphin (AAS18434), Australian lungWsh POMC (AAD37347), African lungWsh POMC (AAD29144). Identical amino acid positionswithin orthologs sequences are shaded. The (¤) indicates proposed endoproteolytic cleavage sites. Opioid sequences are underlined. Abbreviations: ME(met-enkephalin), ME-7 (met-enkephalin 7), ME-8 (met-enkephalin 8), LE (leu-enkephalin), �-Neo (�-neoendorphin), DYN A (dynorphin A), and DYNB (dynorphin B).

310 J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314

amphibians POMC gene is 4.7 million years. Based on thisanalysis it appears that at least the amphibian proenkepha-lin and POMC genes may be evolving at the same rate.Moreover, the UEP values suggest that the amphibianopioid genes are evolving at a faster rate than the lungWshopioid genes.

A similar trend is observed for the mammaliansequences presented in Fig. 4. For calculating UEP values,the divergence times for the rat and human lineages is set at85 million years. The organization of the mammalian pro-enkephalin sequences (Yoshikawa et al., 1984; Noda et al.,1982) is remarkably similar to the lungWsh and amphibian

Fig. 3. Anuran amphibian opioid precursor sequences. The following anuran amphibian opioid precursor amino acid sequences were aligned as describedin Dores et al. (1996): Xenopus laevis Proenkephalin (AAB20686), Bombina orientalis Proenkephalin (AAU951555), X. laevis Prodynorphin (AA084050),B. orientalis Prodynorphin (AA084051), X. laevis POMC (PO6298), B. orientalis POMC (AAU95754).). Identical amino acid positions within orthologssequences are shaded. The (¤) indicates proposed endoproteolytic cleavage sites. Opioid sequences are underlined. Abbreviations: ME (met-enkephalin),ME-7 (met-enkephalin 7), ME-8 (met-enkephalin 8), LE (leu-enkephalin), �-Neo (�-neoendorphin), DYN A (dynorphin A), and DYN B (dynorphin B).

J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314 311

organizational schemes, and the primary sequence identityfor the mammalian proenkephalin sequences is 79%.Hence, the UEP value for the mammalian proenkephalinsequences is estimated to be 4.1 million years, and is similarin duration to the amphibian UEP for proenkephalin.

The mammalian prodynorphin sequences contain a �-neoendorphin sequence, a dynorphin A sequence, and adynorphin B sequence (Takahashi et al., 1981; Civelli et al.,

1985), but lack additional pentapeptide opioid sequences inthe N-terminal region of the precursor. When the fulllength mammalian prodynorphin sequences are compared,the level of primary sequence identity is 56%. This valuedrops to 52% when the analysis is restricted to positions45–225 (Fig. 4). As a result, the UEP value for the mamma-lian prodynorphin genes ranges between 1.8 and 1.9 millionyears. Finally, there appears to be a bit more stability in the

Fig. 4. Eutherian mammal opioid precursor sequences. The following mammalian opioid precursor amino acid sequences were aligned as described inDores et al. (1996): human Proenkephalin (PO1210) rat Proenkephalin (PO4094), human Prodynorphin (NP077722), rat Prodynorphin (AAA41118),human POMC (PO1189), rat POMC (PO1194).). Identical amino acid positions within orthologs sequences are shaded. The (¤) indicates proposed endo-proteolytic cleavage sites. Opioid sequences are underlined. Abbreviations: ME (met-enkephalin), ME-7 (met-enkephalin 7), ME-8 (met-enkephalin 8), LE(leu-enkephalin), �-Neo (�-neoendorphin), DYN A (dynorphin A), and DYN B (dynorphin B).

312 J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314

mammalian POMC sequences (Drouin et al., 1985). Thelevel of primary sequence identity is 66%, and as a result theUEP value for the mammalian POMC genes is estimated at2.6 million years. Hence, the mammalian and anuranamphibian opioid-coding genes display similar rates of evo-lution. In the both groups the proenkephalin and POMCgenes appear to be evolving at the same rates, whereas, theprodynorphin genes in both tetrapod groups appear to beevolving faster than the other paralogs in this gene family.This observation is interesting, in that the prodynorphingene is predicted to be the most recent member of the opi-oid/orphanin gene family (Danielson et al., 2002; Doreset al., 2004).

Based on the observations made for Figs. 2–4, it appearsthat the paralogs in the opioid/orphanin gene family areevolving at relatively the same rate within the lineages con-sidered for this study. However, the orthologs in this genefamily are undergoing lineage speciWc rates of evolution.Hence, it would be reasonable to assume that the genomesof the lineages (i.e., lungWsh, anuran amphibian, and placen-tal mammal) selected for this study are evolving at diVerentrates, and that mutations are accumulating in the lungWshopioid genes at a slower rate than in either the amphibianor mammalian opioid genes. When these observations arecoupled with the earlier studies on the rate of morphologi-cal change and the degree of species diversity over time inthe lungWsh lineage (Simpson, 1944, 1953), it would be rea-sonable to propose that the lungWsh opioid genes may beretaining features that could have been present in the ances-tral sarcopterygian opioid genes. Hence, lungWsh may be agood indicator of how trends in opioid gene evolution haveprogressed in the extant Sarcopterygii.

For example, it does appear that the ancestral organiza-tional plan for the proenkephalin gene in gnathostomes isto encode seven opioid sequences. Among the Sarco-pterygii, only mammalian proenkephalin encodes a leu-enkephalin sequence at the sixth opioid position. Thepresence of a met-enkephalin sequence at this position inlungWsh, amphibians and shark (Khalap et al., 2005) wouldsupport the hypothesis that a met-enkephalin sequence waspresent at the penultimate position in the proenkephalin ofthe ancestral gnathostomes. Given the divergence times forthe stem gnathostome groups [at least 420 million years(Carroll, 1988)], the organization plan for the gnathostomeproenkephalin gene has been remarkably conserved.

By contrast, the prodynorphin gene has undergoneextensive primary sequence changes in the lineages includedin this study. The presence of Wve opioid sequences in boththe lungWsh and anuran amphibian prodyorphin sequencesis consistent with the hypothesis that prodynorphin genearose as a result of the duplication of the proenkephalingene (for review see Khalap et al., 2005). The pentapeptideopioid sequences located in the N-terminal portion of thelungWsh, X. laevis, and B. orientalis prodynorphinsequences is consistent with this hypothesis. Interesting, inthe prodynorphin gene of the anuran amphibian, Bufomarinus only one of the pentapeptide opioid sequences is

present (Danielson et al., 2002). Perhaps some lineages ofanuran amphibians have undergone the trend observed inmammals that has resulted in a reduction of the pentapep-tide opioid sequences in the prodynorphin gene. Anotherfeature in both lungWsh and B. orientalis prodynorphin thatis a reXection of the proenkephalin signature in the prody-norphin gene is the presence of a methionine residue at theWfth position in the dynorphin A sequence (Fig. 2, position224; Fig. 3, position 238). The dynorphin A sequence inprodynorphin can be aligned to the Wfth opioid sequence(met-enkephalin) in gnathostome proenkephalin sequences(Danielson and Dores, 1999), thus, the presence of a methi-onine residue at this position would represent the ancestralcondition for the dynorphin A sequence.

Perhaps the POMC gene is the best indicator that thelungWsh genome has retained ancestral features. The orga-nization plan for the POMC gene is nearly as highly con-served in gnathostomes as the organizational plan for theproenkephalin gene. All gnathostome POMC sequencesencode ACTH (with �-MSH as the N-terminal sequence inACTH) and �-MSH (for review see: Dores and Lecaude,2005). The presence of the �-MSH sequence in the POMCgene of cartilaginous Wsh and in the POMC gene of the Sar-copterygii, coupled with the apparent degeneration of the�-MSH sequence in some ray-Wnned Wshes and the com-plete loss of this sequence in teleosts, would indicate thatthe �-MSH sequence is undergoing secondary loss in theActinoptergyii. However, of greater interest to this discus-sion is the nature of the �-endorphin sequence. The lungWsh�-endorphin sequence presented in Fig. 2 (Y229-Q263) hasthe amino acid motif W237D238 and a glutamate residue atposition 259. These three amino acids are absent from thetetrapod �-endorphin sequences in Figs. 3 and 4, but havebeen found in nearly every ray-Wnned Wsh and some carti-laginous Wsh �-endorphin sequences (Dores and Lecaude,2005). The “WD” amino acid motif appears to be an ances-tral feature of gnathostome �-endorphin that the lungWshPOMC gene has retained.

Certainly the lungWsh genome is not frozen in time.However, there are indications that this genome is evolvingat a slower rate in both time and space as compared to thetetrapod genomes. Based on the preceding observations, itwould be reasonable to propose that other neuropeptideand polypeptide hormone-coding gene families in the lung-Wsh genome have retained a higher degree of ancestral fea-tures than is seen in other sarcopterygian species. Hencefrom a molecular evolution standpoint, the lungWshes doappear to be “living fossils.” While a case can be made forthe lungWshes that there is a correlation between the rate ofmorphological change and stasis in speciation (Simpson,1953; Stanley, 1998), and a slowing of the rate of molecularevolution in these organisms, identifying the mechanismthat would result in a decline in the rate of gene evolutionhas been elusive. Perhaps a comparative approach can leadto a solution. It would be interesting to look, at the molecu-lar level, at trends in the evolution of chemical communica-tion systems in vertebrates that are undergoing relatively

J. Lee et al. / General and Comparative Endocrinology 148 (2006) 306–314 313

rapid morphological change that is accompanied by rapidspeciation events, as is seen in some species of teleosts. Bycomparing reproductive strategies and life cycle patterns,clues to the factors that inXuence the rate of gene evolutionmay emerge.

Acknowledgments

This research has been supported by the following NSFgrants: NSF IBN-9810516, NSF IBN-0132210, and NSFIOB 0516958 (RMD). This research would not have beenpossible without our collaboration with Prof. Jean Joss.She has been the driving force for many of these studies,and she has been a champion for the preservation of theAustralian lungWsh habitat.

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