Morphological, Ecological, and Molecular Analyses Separate Muraenaaugusti from Muraena helena as a Valid Species

S. JIMENEZ, S. SCHONHUTH, I. J. LOZANO, J. A. GONZALEZ, R. G. SEVILLA, A. DIEZ, AND

J. M. BAUTISTA

A multidisciplinary approach including biometric, ecological, and molecular genetic

analyses was employed to test species status of the Black Moray Muraena augusti (Kaup,

1856) and the Mediterranean Moray M. helena Linnaeus, 1758. Relevant differences

were identified in their habitat, bathymetric range, color pattern, vertebral formula,

growth parameters and lifespan, reproduction strategy, size/age at first maturity, and

distribution. Intra- and interspecific genetic divergences (based on a region of the

rhodopsin gene and the complete cytochrome b gene) also suggest that both moray eels

are different species. Mitochondrial and nuclear sequence data place these two species

into different clades. Phylogenetic analyses among an additional five species of moray

eels occurring in sympatry in the eastern central Atlantic resolved M. helena, M. augusti,

and M. melanotis as closely related species in a well supported clade, while M. robusta

emerged as a more divergent species within the Gymnothorax clade. Based on these

findings, Muraena augusti is a valid species and should be resurrected.

THE best-known species of the genus Muraenais the Mediterranean Moray (Muraena he-

lena). This subtropical, reef-associated specieswas first described by Linnaeus in 1758 andoccurs across the eastern Atlantic from south ofthe British Isles to Senegal, including theMediterranean, the Azores, Madeira, the Canary,and Cape Verde Islands (Bohlke, 1981; Bauchot,1987; Brito et al., 2002). The Black Moray fromthe eastern central Atlantic was described on thebasis of type material from Madeira as Thryrsoideaaugusti (Kaup, 1856). Until the mid 20th century,both species were considered valid, although T.augusti had earlier been reassigned to Muraenaaugusti by many authors (Gunther, 1870; Maul,1948; Albuquerque, 1954–1956).

Blache (1967) argued that M. augusti wasa junior synonym of M. helena. Since then, severalreviews, the Eschmeyer classification (http://www.calacademy.org/research/ichthyology/catalog)and regional catalogs accepted this synonymy(Blache et al., 1973; Bauchot, 1986; Smith andBohlke, 1990). Some authors, however, maintainthat the two forms are valid species (Bohlke et al.,1989; Jimenez, 1997; Brito et al., 2002). In recentyears, some of these authors have pointed out thatM. augusti has an insular distribution restricted tothe Azores, Madeira, Salvages, Canary, and CapeVerde archipelagos (Bohlke, 1981; Brito et al.,2002).

The current classification of the moray eels ismainly based on the work done by muraenidanatomists during the last few decades thatdistinguished muraenid genera and subfamilies,notably the work on gill arches (Nelson, 1966),

branchial muscles (Nelson, 1967), and discussionof muraenid relationships (Bohlke et al., 1989).Concerning the moray eels from the easternAtlantic (80uN–23uS), it can be summarized thatpresence of rudimentary dorsal and anal fins areused to distinguish Anarchias, Channomuraena,and Uropterygius (subfamily Uropterygiinae) fromthe remaining moray eels (subfamily Muraeni-nae). Within Muraeninae, mostly blunt, molar-like teeth separate Echidna from the othergenera. Large mouth cleft and dorsal-fin origin(above or behind gill opening) are used todistinguish Enchelycore. Finally, presence of pos-terior and anterior nostrils separate Muraenafrom Gymnothorax species (Bohlke, 1981; Bau-chot, 1986).

Morphological similarities between M. augustiand M. helena include body shape, tubularposterior and anterior nostrils, non-serratedteeth along their anterior and posterior margins,number of head pores, and number of predorsalvertebrae. In contrast, morphological differencesinclude upper jaw teeth mostly biserial, lower jawteeth biserial anteriorly (in smaller specimens),uniserial posteriorly (in larger specimens of M.augusti), all jaw teeth uniserial (in M. helena),number of preanal and total vertebrae, colorpattern, habitat and bathymetric range, andgeographical distribution. Through the exami-nation of extensive material from the Canarieswhere both zoological forms live in sympatry, thepresent report describes a multidisciplinary ap-proach including biometric, biological, andmolecular genetic analyses designed to establishwhether both forms represent different species.

Copeia, 2007(1), pp. 101–113

# 2007 by the American Society of Ichthyologists and Herpetologists

MATERIALS AND METHODS

Biometric analyses.—Between 1985 and 1994, 434specimens of M. augusti and 750 of M. helena wereobtained from commercial catches at differentfishing ports in the Canary Islands. Specimenswere caught with bottom traps, handlines, andbottom longlines (Jimenez et al., 1993) at depthsof 2–128 m (M. augusti) and 1–801 m (M.helena). Collected morays were assigned to eachspecies according to their different color pattern.Dark purplish-black specimens, with minute,widely-separated, light spots on the body andfins, and striking white eyes were assigned to M.augusti (Fig. 1A). Brown specimens, with largepale yellowish spots each of them containingsmaller brown spots, forming a ‘‘rosette’’ (paleareas becoming smaller posteriorly with fewerbrown spots, and sometimes the end of tail haswhite blotches), head brown with several paleyellowish spots and reticulations, gill openingsand corner of mouth black-edged, and fins witha light margin were assigned to M. helena(Fig. 1B).

Total length (TL), preanal length (PA), headlength (HL), snout length (S), snout to rictus( J), eye diameter (E), and interorbital width(IOW) were measured to the nearest mm. Thenumber of supraorbital (SO), infraorbital (IO),mandibular (M), and branchial pores (B) werecounted in each specimen. A subsample of nineindividuals from both M. augusti and M. helena

were taken to establish the vertebral formula byrecording predorsal (PD), preanal (PA), andtotal numbers of vertebrae (T) using radio-graphs. Individuals were selected using a randomlength stratified method, which divided speci-men total length into three wide size classes(small, medium, and large) and selected individ-uals in accordance to the relative abundance ofeach class with respect to the whole sample ofeach species. All measurements and counts ofpores and vertebrae as well as their abbreviationsfollow Bohlke (1982) and Bohlke et al. (1989),except for IOW.

Total body weight (TW) of each eel wasmeasured to the nearest g. Sex and maturitystages were then determined macroscopicallyand gonad weight (GW) was recorded to thenearest 0.1 g. Stages of maturation were classifiedas follows: I, immature; II, resting; III, ripe; IV,running ripe; and V, spent following Holden andRaitt (1975). Otoliths were removed from 296Black Morays and 225 Mediterranean Morays,cleaned and stored dry. Age was determinedfrom right otoliths placed in glycerine andexamined under a compound light microscopewith reflected light against a dark background.Left otoliths were embedded in plastic resin toobtain thin sections (0.4 mm; Bedford, 1983).Each otolith was read twice and only equalreadings were accepted. The von Bertalanffygrowth function (VBGF) was fitted to theobserved length-at-age data of the resulting age-

Fig. 1. (A) Muraena augusti, TFMC BMVP/01443, 782 mm TL; (B) Muraena helena, TFMC BMVP/00884,825 mm TL.

102 COPEIA, 2007, NO. 1

length key using Marquardt’s algorithm for non-linear least-square parameter estimation (Pauly,1983). The form of the growth curve followingBeverton and Holt (1959) is:

Lt ~ L? 1 { e{k t { toð Þ� �

,

where Lt is fish length (mm TL) at time t (year), L‘

(mm TL) the asymptotic length to which fish tendto grow, k (year21) the growth coefficient, and t0

(year) the hypothetical time when fish length iszero. Lifespan A0.95, the age at which the fishreaches 95% of its asymptotic length, was alsocalculated (Taylor, 1959). The spawning seasonwas determined according to monthly changes inthe gonadosomatic index (GSI), calculated asfollows (Anderson and Gutreuter, 1983):

GSI ~ 10n GW=TW,

where n is a coefficient conveniently chosen toreduce the number of decimals.

For the estimation of mean length at 50%

maturity, a logistic function was fitted to theprobability of the mature individuals by size classusing a non-linear regression. The function usedwas (Pope et al., 1983):

Pi ~ 1.

1 z e{ a z b Lið Þ� �

,

where Pi is the probability of mature individualsin each size class (Li), and a and b, theparameters to be estimated.

Genomic analysis.—Our preliminary analyses of16S, including six genera of the subfamilyMuraeninae, suggest Gymnothorax and Muraenaare closely related genera (unpubl. data). Forcomparison to specific divergences between M.augusti and M. helena we included five additionalclosely related species that occur in sympatry inthe eastern Atlantic with the two putative species.Genetic divergences and phylogenetic relation-ships were assessed by analyzing 24 specimens ofmoray eels from different populations of Mur-aena augusti, M. helena, M. melanotis, M. robusta,Gynmothorax unicolor, G. maderensis, and G. afercollected in the eastern Atlantic and the Medi-terranean. The complete mitochondrial cyto-chrome b gene and a fragment of the rhodopsinnuclear gene were sequenced in all specimens,representing seven species from 12 differentsample sites. For each species, two voucherspecimens of total DNA, muscle tissue andotoliths were deposited in the biological refer-ence collections of the Museo de CienciasNaturales de Tenerife and Instituto do Mar–Natural History Museum of Funchal.

DNA extraction and PCR amplification.—Total DNAwas extracted from muscle tissue according tostandard proteinase K/phenol-chloroform pro-tocols (Sambrook et al., 1989). For the cyto-chrome b gene, combinations of eight versatileprimers were used, six of which were used innested PCR amplifications to isolate two contig-uous and overlapping fragments (of 685 bp and667 bp) covering the entire cytochrome b gene.For Muraena melanotis we designed new primersto obtain the second part of the cytochromeb gene (MMB-F and MMB-R) due to amplifica-tion difficulties with previous primers (Table 1).Nested PCR amplification was conducted usingfour versatile primers to isolate a 515-bp frag-ment of the rhodopsin gene (Table 1).

The 25 ml PCR reaction mixtures contained:2.5 ml 103 ampliTaq Gold buffer, 2 mM MgCl2,0.4 mM dNTPs, 0.4 mM of each primer, 2 ml DNAextraction, and 1 unit Taq DNA polymerase(ampliTaq Gold, Applied Biosystems). After aninitial denaturation step at 94 C for 7 min, 40cycles were performed as follows: denaturation at94 C (30 sec), annealing at 50 C (30 sec; forcytochrome b) or 62 C (30 sec; for rhodopsin),and extension at 72 C (30 sec), with a finalextension of 7 min. For the second nested PCR,1 ml of the first PCR product was used astemplate. PCR conditions were identical to firstPCR except annealing (52 C for cytb and 60 Cfor rhodopsin). Both strands were sequencedusing primers of the second nested PCR ampli-fication using an automated DNA sequencer(Applied Biosystems 3730). GenBank accessionnumbers are listed in Material Examined.

Sequence analyses.—Twenty-four complete cyto-chrome b gene sequences (1140 bp) and 25rhodopsin sequences (515 bp) were alignedmanually with outgroup sequences of Congermyriaster (NC002761, AB038381) and Anguillaanguilla (AF006715, L78007). No ambiguousalignments or gaps were found. All codonpositions were included in the analyses. Geneticdivergence percentages are based on uncorrect-ed p-distances. Three different methods ofinferring phylogenies to assess congruence be-tween methods were used (Schonhuth andDoadrio, 2003). Phylogenetic trees were estimat-ed using Maximum Parsimony (MP), MinimumEvolution (ME), and Bayesian Inference (BI).Maximum parsimony analyses involved heuristicsearches with ten random stepwise additions oftaxa, save multiple tree option and tree bisection-reconstruction branch swapping. Transversions(Tv) were weighted six times over transitions (Ts)for the cytochrome b gene, two times the weightof Ts for the rhodopsin gene, and four times the

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 103

weight of Ts for the combined data set based onempirical evidence. The hierarchical likelihoodratio test (hLRT) implemented in MODELTESTV3.4 (Posada and Crandall, 1998) was used tofind the evolutionary model that best fitted thedata, and optimized parameter values were usedto estimate ML distances for ME analyses.

All analyses were performed with three differ-ent nucleotide sequence datasets, completemitochondrial cytochrome b sequences, partialnuclear rhodopsin sequences, and a combinedCytb + Rhod gene data set. Phylogenetic analyseswere conducted with PAUP* (vers. 4.0b10, D. L.Swofford, Sinauer Associates, Inc., Sunderland,MA, 2001) and MrBayes v3.0 (Huelsenbeck andRonquist, 2001). The robustness of the inferredtrees was tested by bootstrapping (Felsenstein,1985), performing 1,000 pseudoreplications forMP and ME. For BI 1,000,000 generations wererun sampling the Markov chain at intervals of 100generations. A total of 1,000 out of 10,000resulting trees were discarded as ‘‘burn-in.’’Support for tree nodes was determined basedon the values of Bayesian posterior probabilityobtained from a majority-rule consensus tree.

The deduced amino acid sequences for boththe nuclear and mitochondrial gene codedproteins were combined into a single data setusing MEGA v 2.1 (S. Kumar, K. Tamura, I. B.Jacobsen, and M. Nei, Arizona State University,Tempe, AZ, 2001) and then analyzed in PAUP*.Minimum evolution analyses for amino acidsequences were based on mean character differ-ences.

RESULTS

Color patterns.—The morays studied exhibited thespecific color pattern as described in Materialsand Methods, i.e. ‘‘black’’ pattern for M. augustiand ‘‘marbled’’ pattern for M. helena. No mixedor intermediate color patterns were found.

Morphometric and meristic characteristics.—Morpho-metric characteristics (Table 2A), head porefrequencies (Table 2B), and vertebral frequen-cies (Table 2C) were obtained for both M.augusti and M. helena. Among these character-istics, the most distinctive was found in vertebralfrequencies for both species that did not overlapin preanal and total vertebrae. Muraena augustiranged in size from 362 to 900 mm TL, weighingbetween 75 and 1698 g, while M. helena from 419to 1340 mm TL and weighing between 109 and6450 g.

Age and growth.—Length-weight relationshipswere described by the following parameters: a 5T

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104 COPEIA, 2007, NO. 1

4.48 3 1027, b 5 3.2466, r2 5 0.9258 (t-test, t 5

5.572 . t0.01, df 5 431 5 2.576) for M. augusti; a 5

2.47 3 1027, b 5 3.3141, r2 5 0.8876 (t-test, t 5

7.277 . t0.01, df 5 747 5 2.576) for M. helena.Positive allometric growth was observed for bothmorays (P , 0.01).

When the otoliths were covered with glycerine,growth rings in otolith sections were clear. For M.augusti, 96% of whole otoliths and 78% of otolithsections were readable. For M. helena these valueswere 88% and 89%, respectively. The generalpattern indicated that two rings, one opaque andone hyaline, are deposited over a single year inboth species.

Muraena augusti was aged 4–12 years (Table 3).Parameters of the VBGF were: L‘5 1059 mm TL,k 5 0.178 year21, t0 5 1.103 year (n 5 90, 410–890 mm TL, r2 5 0.9306). Lifespan was A0.955

17.9 years. Muraena helena was aged 3–15 years(Table 4). Parameters of the VBGF were: L‘5

1700 mm TL, k 5 0.078 year21, t0 5 20.355 year(n 5 179, 490–1230 mm TL, r2 5 0.9054).Lifespan was A0.95 5 38.1 years.

Sexuality and reproduction.—The overall ratio ofmales to females was 1:1.45 for M. augusti and1:1.38 for M. helena. No macroscopic evidence ofhermaphroditism was found when examining thegonads of both types of moray, which appeared

to be gonochoric. In M. augusti, the highest GSIvalues for females and males combined wererecorded between May (10.2 6 7.6) and October(10.3 6 13.1), peaking in August (80.2 6 39.1).For M. helena, the highest GSIs values observedwere from January (19.5 6 13.1) to July (68.0 6

63.7), with a peak in May (103.6 6 22.0). Sexualmaturity parameters were: a 5 210.2598, b 5

0.0183 (n 5 9, r2 5 0.8125, P , 0.001) for M.augusti, and a 5 213.4480, b 5 0.0179 (n 5 16, r2

5 0.8276, P , 0.001) for M. helena. The meanlengths at 50% maturity were 558 mm TL(5.3 years) and 751 mm TL (7.1 years) for M.augusti and M. helena, respectively.

Genetic analyses.—The complete cytochromeb gene and 515 bp of the rhodopsin gene weresuccessfully amplified and sequenced in 24 and25 specimens, respectively, representing fourspecies of the genus Muraena and three speciesof the related genus Gymnothorax.

Of the 1140 bp aligned for cytochrome b, 669sites were invariant and 412 were parsimony-informative. The nucleotide composition of thecytochrome b gene (A: 0.25583, C: 0.28776, G:0.15704, T: 0.29937) indicates a low frequency ofguanine. However, the null hypothesis of fre-quency homogeneity among bases across taxawas not rejected. The frequency of G was

TABLE 2. MORPHOLOGICAL AND MERISTIC CHARACTERS OF THE BLACK AND MEDITERRANEAN MORAY EELS.

(A) Morphometric characteristics (Black Moray n 5 434, size range 5 362–900 mm TL; Mediterranean Moray n 5

749, size range 5 419–1340 mm TL). Variables are expressed as a percentage of the total length (TL) and headlength (HL).

Moray

TL HL

PA HL S J E IOW

Black 38.9–52.0 9.8–16.2 13.9–27.7 50.0–78.4 5.3–11.9 5.2–14.5Mediterranean 43.4–54.8 9.5–15.8 13.6–25.6 40.6–86.0 4.3–10.5 5.2–14.2

TL: Total length; PA: Preanal length; HL: Head length; S: Snout length; J: Maxilar length; E: Eye diameter; IOW: Interorbital distance.

(B) Head pore frequencies

No. of pores

SO IO M B

1 2 3 4 5 6 4 5 6 7 2 3

Black 12 178 1 189 1 — 4 172 14 1 162 1Mediterranean 3 115 — 126 — 3 1 115 13 — 104 1

SO: Supraorbital pores; IO: Infraorbital pores; M: Mandibular pores; B: Branchial pores.

(C) Vertebral frequencies

Vertebrae

PdV PaV TV

4 5 51 52 53 54 55 59 60 61 62 63 137 139 142 143 144 145 146 148

Black 2 7 2 2 1 3 1 — — — — — 2 7 — — — — — —Mediterranean 2 7 — — — — — 1 2 3 2 1 — — 1 3 1 1 2 1

PD: Predorsal vertebrae; PA: Preanal vertebrae; T: Total vertebrae.

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 105

particularly low in third codon positions (G:0.09558) as also occurs in cyprinids (Bielawskiand Gold, 1996).

Of the 515 bp aligned for rhodopsin, 354 siteswere invariant and 95 were parsimony-informa-tive. The nucleotide composition of the rhodop-sin gene region (A: 0.19078, C: 0.31528, G:0.24338, T: 0.25056) indicates a low frequency ofadenine, as also detected in other Teleostei(Chen et al., 2003), which is particularly evidentin third codon positions (A: 0.09387). However,the null hypothesis of the homogeneity test ofbase frequencies across taxa was not rejected. Forthe combined data set (1656 bp) 1024 sites wereinvariant and 506 were parsimony-informative.

Pairwise sequence divergences (uncorrected p-distances) among the muraenid species of thegenus Muraena analyzed ranged from 13.1–21.1%

for cytochrome b and 1.7–6.5% for rhodopsin(Table 5). Divergences between the generaMuraena and Gymnothorax ranged from 17.0–21.5% for cytochrome b and 4.6–7.8% forrhodopsin. Divergences among geographic sam-ples of M. helena were 0.2–0.8% for cytochrome b,whereas no polymorphisms were detected in

rhodopsin sequences. Divergences between spe-cimens of M. augusti ranged from 0.0–1.6% inthe cytochrome b gene and 0.0–0.6% for rho-dopsin. Genetic divergences between both puta-tive moray eel species were in the range of 1.5–2.1%, based on the rhodopsin gene, and 14.7–15.6%, according to the cytochrome b gene(Table 5).

The three methods of phylogenetic inferences(MP, ME, and BI), recovered congruent topolo-gies. Nuclear and mitochondrial DNA alwaysdistinguished between M. helena and M. augusti,using Conger myriaster and Anguilla anguilla asoutgroups. However, the two genes produceddifferent topologies (Figs. 2–3). All trees placedM. helena and M. augusti into two independentclades corresponding to putative species designa-tions. Muraena melanotis was closely related tothese species, but its phylogenetic position variedbetween genes. In contrast, M. robusta alwaysclustered with G. maderensis and G. unicolor in thecytochrome b tree, but held a more basal positionin the rhodopsin tree (Figs. 2–3). The largestdivergences for the cytochrome b gene wererecorded between the pairs M. augusti and M.

TABLE 3. AGE-LENGTH KEY FOR THE BLACK MORAY.

Size (mm TL)

Age group (year)

IV V VI VII VIII IX X XI XII

410 2430 1450 3 1470 2 3490 5510 3530 5550 2 4570 2 3590 7610 7630 1 8650 2 4670 4690 1710 1730 4750 1 1770 3790 1 1810 3830 1 1850 1870 1890 1n 8 22 31 15 5 5 2 1 1x 445 518 602 691 769 811 842 873 890sd 27 41 29 34 15 14 11 0 0

106 COPEIA, 2007, NO. 1

robusta (20.3–21.1%), M. augusti and G. unicolor(20.2–21.2%), and G. afer and M. robusta (21.2–21.5%), and were similar to those found betweenA. anguilla and C. myriaster (20.8%), or betweenA. anguilla and moray eel species (19.9–22.6%).However, when divergences were considered inthe rhodopsin gene sequence, values were muchlower between pairs of muraenid species (1.5–7.8%) than between muraenid species and A.anguilla (15.8–17.8%) or C. myriaster (19.8–21.3%). Phylogenetic analyses of the combinednucleotide (1656 bp) and amino acid (551

positions: 455 invariant and 41 parsimony-in-formative) datasets also grouped M. augusti andM. helena in two independent clades closelyrelated to M. melanotis (trees not shown).

DISCUSSION

Muraena augusti and M. helena differ in colorpattern, morphology, ecology, and biologicalaspects (age, growth, and reproduction), as wellas in mitochondrial and nuclear DNA sequences.Black and Mediterranean Morays exhibit typical

TABLE 4. AGE-LENGTH KEY FOR THE MEDITERRANEAN MORAY.

Size(mmTL)

Age group (year)

III IV V VI VII VIII IX X XI XII XIII XIV XV

490 1510 1530 1550 1 1570 1 2590 3610 1630650 3670 2690 1 2710 2730 8750 3 4770 2 6790 1 7810 1 2 3830 6 1850 1 13870 5 14890 1 8 5910 8 1930 5950 2 10 2970 1 3 2990 1 1 2 2 11010 1 1 21030 1 1 1 31050 21070 1 11090 11110 1 1 11130 1 11150 11170 11190 1 112101230 1n 1 1 4 7 24 32 43 35 9 5 13 3 2x 485 544 539 597 726 809 872 944 978 1065 1059 1143 1220sd 0 0 31 39 49 41 37 40 35 84 49 25 28

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 107

‘‘black’’ and ‘‘marbled’’ patterns respectivelyalong their size, bathymetric, and geographicranges. The vertebral formula for the two morayswas found to differ, with M. augusti having lowerranges of preanal and total vertebrae than M.helena. Snout length in M. augusti is slightlylonger than that of the M. helena.

In the Canary Islands, M. augusti is a nocturnalterritorial species inhabiting rocky and stonybottoms, sometimes hiding under large rocks intidal pools, from shore to a depth of 250 m,mainly shallower than 50 m. Muraena helena isa territorial species found on rocky bottoms,commonly lurking in holes and writhing throughcrevices or under rocks, from shore to a depth of800 m; rarely or infrequently shallower than50 m and commonly at depths of 100–300 m(Franquet and Brito, 1995; Jimenez, 1997; Britoet al., 2002). Specimens of M. augusti examinedwere aged between 4 and 12 years, their asymp-totic length was a little over 1 m, growth co-efficient estimated to be 2.28 times higher thanthat of the M. helena, and lifespan has beenestimated to be 50% of that of M. helena.Specimens of M. helena examined were aged 3to 15 years; their asymptotic length was estimatedat 1.7 m TL, growth coefficient very slow, andlifespan very long. The reproduction period forM. augusti in the Canaries runs from May toOctober, with a peak in August; its size atmaturity was estimated at 558 mm (5.3 yearsold). Muraena helena spawn between Januaryand July, with a peak in May, with size at maturityat 751 mm (7.1 years). Muraena augusti areknown to be endemic from the Macaronesianarchipelagos, whereas M. helena show a wideAtlantic–Mediterranean distribution pattern in-cluding both continental and insular coasts fromthe British Isles to Senegal. Based on all thesedifferences between both morays, it is reasonableto consider them as distinct species.

Molecular genetic analyses performed on in-dependent data from different genomes (mito-chondrial cytochrome b gene and nuclear rho-dopsin gene region) in both moray forms, plusfive sympatric related species was congruent withthe morphological analyses. Intraspecific se-quence divergences in both moray eels werebelow 1.6% for the complete cytochrome b gene,while interspecific sequence divergence betweenboth morays species was between 14 and 70 timesthe intraspecific values. This divergence, in therange 14.7 to 15.6%, is similar to that foundamong species of the genus Muraena (13.1–21.1%) or between species of the genera Muraenaand Gymnothorax (17.0–21.5%). These divergencevalues are greater than those observed betweenother species pairs of the genus Anguilla (4.3–

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108 COPEIA, 2007, NO. 1

Fig. 2. Minimum evolution tree showing phylogenetic relationships among moray eel geographicalsamples and species analyzed based on cytochrome b sequence data. Minimum evolution bootstrap andBayesian posterior probabilities support above branches and MP bootstrap values (.50%) are indicatedbelow branches. Terminal labels represent species and locality. CI: Canary archipelago, MA: Madeiraarchipelago, WM: Western Mediterranean, EE: Mauritania coast.

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 109

Fig. 3. Minimum evolution tree showing phylogenetic relationships among moray eel geographicalsamples and species analyzed based on rhodopsin sequence data. Minimum evolution bootstrap andBayesian posterior probabilities support above branches and MP bootstrap values (.50%) are indicatedbelow branches. Terminal labels represent species and locality. CI: Canary archipelago, MA: Madeiraarchipelago, WM: Western Mediterranean, EE: Mauritania coast.

110 COPEIA, 2007, NO. 1

10.4%) for the same gene (Aoyama et al., 2001).Intraspecific divergences for 515 bp of therhodopsin gene were below 0.6% while diver-gences observed between both moray eel speciesranged from 1.5–2.1%, comparable to the rangeobserved between M. augusti and M. melanotis(1.7–2.1%). This dataset provides appreciabledifferences in genetic divergences between close-ly related species considering nuclear vs. mito-chondrial genes. Given that mitochondrial genesevolve faster than nuclear DNA (Vawter andBrown, 1986), these data can serve as the basisfor future studies in this group, suggesting thecollapse of anguilliform basal relationships inwide frame anguilliform phylogenies based oncytochrome b because of the saturation effect offast evolving genes (Moriyama and Powell, 1997).In contrast, the rhodopsin gene could lead tohigher resolution in these basal nodes.

The present study provides the first molecularphylogenetic hypothesis among seven species ofeastern Atlantic morays. These results indicatethat M. augusti is a valid species, separated fromM. helena and with M. melanotis as a closely relatedspecies. Moreover, mitochondrial and nuclearDNA data indicate that M. robusta is closer toGymnothorax than to the remaining species of thegenus Muraena included here. Further work onmore taxa should serve to establish phylogeneticrelationships across a wider range of moray eels.

A recent study on population maintenance intropical reef fishes (Robertson, 2001) suggestedthat most endemics belong to regionally species-rich families. However, Robertson listed mem-bers of the family Muraenidae as exceptions, asthey are regionally species rich, but lack islandendemics. This may be explained by their longpelagic larval interval, which is among thelongest known for reef fishes (Hourigan andReese, 1987). In addition, the long distancedispersal of moray larvae could reduce thelikelihood that island populations are sufficientlygenetically isolated for endemics to develop.Muraena helena has a wider geographic rangeextending to Mediterranean waters with littlegenetic divergence among specimens from dif-ferent localities (0.2–0.8%), while M. augusti,restricted to the eastern central Atlantic Ocean,shows greater intraspecific genetic divergence(0–1.6%). The overlapping geographical distri-bution and similar morphological characters ofM. helena and M. augusti (Jimenez, 1997) wouldexplain why some authors consider these twospecies as synonymous. Data reported here,nevertheless, reveal a large number of differ-ences in morphological, biological, and genetictraits that establish M. augusti as a differentspecies. Black Morays, therefore, should be

reassigned to Muraena augusti (Kaup, 1856) andconsidered separate from the MediterraneanMoray Muraena helena Linnaeus, 1758.

MATERIAL EXAMINED

Institutional abbreviations follow Leviton et al.(1985), except for the following: TFMC 5

Tenerife Museum of Natural Sciences, CanaryIslands. Code of extracted tissue, locality, collec-tion number of voucher specimens, and Gen-Bank accession numbers (cyt b and rhodopsin),are as follows: Muraena helena: Mhel01CI, Bar-ranco del Binto, El Hierro, Canary Islands, TFMCBMVP/0496, AY862092, AY862115; Mhel02CI,Barranco del Binto, El Hierro, Canary Islands,no voucher, AY862063, AY862116; Mhel6IB, nospecific locality data, Ibiza (Balearic Islands), novoucher, AY862096, AY862117; MurHelWM02,Santa Pola, Alicante, TFMC BMVP/0884,AY862094, AY862118; MurHelMa05a, no specificlocality data, Madeira Island, no voucher,AY862095, AY862119. Muraena augusti: Maug01CI,La Restinga, El Hierro, Canary Islands, TFMCBMVP/0500, AY862097, AY862107; Maug02CI, LaRestinga, El Hierro, Canary Islands, no voucher,AY862098, AY862108; MAU01CI, no specific local-ity data, Canary Islands, no voucher, AY862100,AY862110; MAU02CI, no specific locality data,Canary Islands, no voucher, AY862101, AY862111;MAU03CI, no specific locality data, Canary Islands,no voucher, AY862102, AY862112; MAU04CI, nospecific locality data, Canary Islands, no voucher,AY862103, AY862113; MAU05CI, no specific local-ity data, Canary Islands, no voucher, AY862104,AY862114; MurAugMA01a, no specific localitydata, Madeira Island, MMF-35587, AY862099,AY862109. Muraena melanotis: Mmel01EEa, nospecific locality data, Mauritania, TFMC BMVP/1100, AY862090, AY862120; Mmel02EEa, no spe-cific locality data, Mauritania, no voucher,AY862090, AY862120. Muraena robusta: Mrob01EE,no specific locality data, Mauritania, TFMCBMVP/1267, AY862106, AY862122; Mrob02EE,no specific locality data, Mauritania, TFMCBMVP/1276, AY862105, AY862123. Gymnothoraxunicolor: Guni01CI, Tazacorte, La Palma, CanaryIslands, TFMC BMVP/0498, AY862085, AY862130;Guni02CI, La Restinga, El Hierro, Canary Islands,no voucher, AY862086, AY862131; GuniMA01, nospecific locality data, Madeira Island, MMF-35276,no accession number, AY862128; GuniMA04, nospecific locality data, Madeira Island, no voucher,AY862087, AY862129. Gymnothorax afer: Gafer01EE,no specific locality data, Mauritania, TFMC BMVP/1271, AY862089, AY862124; Gafer02EE, no specificlocality data, Mauritania, TFMC BMVP/1275,AY862088, AY862125. Gymnothorax maderensis:

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 111

Gmad01CI, Punta del Mirlo, El Hierro, CanaryIslands, TFMC BMVP/0492, AY862083, AY862126;Gmad02CI, Punta del Mirlo El Hierro, CanaryIslands, no voucher, AY862084, AY862127.

ACKNOWLEDGMENTS

Sebastian Jimenez and Susana Schonhuthcontributed equally to this work. We are in-debted to S. Perez-Benavente for skillful techni-cal assistance. Thanks are also due to F.Hernandez, J. Santana, M. Garcıa, V. Tuset, J.Gonzalez, J. Quiles, and A. Perez for theirlongstanding support and encouragement onthis study, K. Conway for his comments, and L.Ruber and D. Davis for critical reading of themanuscript. Financial support was receivedthrough the FEDER program of the MCyT-Spain(1FD97-1235-C04 MAR and CAL01-020-C3) andEuropean Commission (FishTrace contract,QLRI-CT-2002-02755). Sequencing was per-formed at the DNA Sequencing Core Facility ofthe UCM.

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(SJ) MUSEO DE CIENCIAS NATURALES, OAMC CA-

BILDO DE TENERIFE, SPAIN; (SS, RGS, AD, JMB)DEPARTAMENTO DE BIOQUIMICA Y BIOLOGIA MO-

LECULAR IV, FACULTAD DE VETERINARIA, UNIVER-

SIDAD COMPLUTENSE DE MADRID, MADRID, SPAIN;(IJL) DEPARTAMENTO DE BIOLOGIA ANIMAL,UNIVERSIDAD DE LA LAGUNA, TENERIFE, SPAIN;AND ( JAG) DEPARTAMENTO DE BIOLOGIA PES-

QUERA, INSTITUTO CANARIO DE CIENCIAS

MARINAS, TELDE, GRAN CANARIA, SPAIN. PRESENT

ADDRESS (SS): DEPARTMENT OF BIOLOGY, SAINT

LOUIS UNIVERSITY, SAINT LOUIS, MISSOURI 63103.E-mail: (SJ) chano@museosdetenerife.org; (SS)susana.schonhuth@gmail.com; (IJL) ilozano@ull.es; ( JAG) solea@iccm.rcanaria.es; (RGS)rsevilla@vet.ucm.es; (DA) adiez@vet.ucm.es;and ( JMB) jmbau@vet.ucm.es; Send reprintrequests to JB. Submitted: 29 Dec. 2005.Accepted: 4 Oct. 2006. Section editor: D. Buth.

JIMENEZ ET AL.—MURAENA AUGUSTI VALIDITY 113