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BULLETIN OF MARINE SCIENCE 50(3) 508-5191992 CORAL REEF PAPER

BIOCHEMICAL AND MORPHOMETRIC ANALYSES FORPHYLOGENIC RELATIONSHIPS BETWEEN SEVEN

SNAPPER SPECIES (SUBFAMILY LUTJANINAE) OF THEWESTERN ATLANTIC

Seinen Chow and Patrick J Walsh

ABSTRACT

Fourteen snapper species belonging to the three genera (Lutjanus and two mono typic generaOcyurus and Rhomboplites) of the subfamily Lutjaninae have been described in the westernAtlantic Electrophoretic (on 25 enzyme loci) and skull morphometric (by analysis of varianceand discriminant analysis) comparisons among seven species of the three genera were per-formed Average Neis genetic distances were 0566 plusmn 0207 among five species of Lutjanus0687 plusmn 0148 between five species of Lutjanus and O chrysurus 0869 plusmn 0224 betweenfive species of Lutjallus and R aurorubens and 0877 between O chrysurus and R aurorubensCluster and additive tree analyses based on the genetic distance indicated that I) there areat least two distinct groups (gray and red snapper groups) within the genus Lutjanus 2) thelane snapper (L synagris) has a closer relationship with the red snapper group (L analis andL vivanus) than with the gray snapper group (L apodus and L griseus) and 3) there is acloser relationship between Lutjanus and O chrysurus than between R aurorubens andLutjanus or O chrysurus On the other hand the skull morphometric analysis indicated thatI) the lane snapper (L synagris) has greater affinity with the gray snapper group than withL analis (red snapper) and 2) although O chrysurus has some affinities with R aurorubensthis monotypic genus showed much less distance than R aurorubells from the Lutjanus group

The subfamily Lutjaninae contains six genera Three (Lutjanus and two mono-typic genera Ocyurus and Rhomboplites) have representatives in the western At-lantic ocean where fourteen species have been described (Allen 1985 Anderson1986) These three genera are defined on the basis of 1) skull morphology 2)presence or absence of pterygoid teeth 3) basisphenoid shape 4) number of gillrakers on lower limb and 5) number of spinous dorsal finrays (Evermann andMarsh 1900 Anderson 1986) Evermann and Marsh (1900) suggested separationof Rhomboplites aurorubens from species of Lutjanus mainly because of the cranialdifferences They also described numerous minor morphological peculiarities inOcyurus chrysurus where the skull morphology is also notably different fromspecies of Lutjanus These peculiarities have convinced some systematists toconsider Ocyurus and Rhomboplites as monotypic genera Based on the osteo-logical 0bserva tions Vergara (1980) also described O chrysurus and R aurorubensto be highly apomorphic forms in the subfamily Within the genus Lutjanus onthe other hand Rivas (1966) proposed three groups based on external featuressuch as meristic counts and coloration in which L griseus L synagris and Lanalis are the representatives for each group (ie gray lane and red snappersrespectively) Vergara (1980) also proposed the same three groups according toosteological observations suggesting that the L griseus group is the least distantfrom the incipient form of this genus because of fewer apomorphic charactersand that the L griseus and L mahogoni (same as L synagris group in Rivas)groups are plesiomorphic sister groups of the more apomorphic L vivanus group(same as L analis group in Rivas)

Thus although considerable efforts for the systematics in this animal grouphave been performed (see Allen 1985 for review) the phylogenetic relationships

508

CHOW AND WALSH PHYLOGENETIC STUDY OF ATLANTIC SNAPPER 509

between species ofthe different genera or between species within the genus Lutja-nus are still unclear

For biochemical systematic purposes we have collected five species in the genusLutjanus and two in the two monotypic genera Parallel analyses of enzymeelectrophoresis and skull morphometry were performed for these seven speciesIn this paper we report the results and discuss the phylogenetic relationshipsamong the three genera and the seven species

MATERIALS AND METHODS

Seven snapper species used in this study (Table I) were mostly caught in Miami or Key West areasA few of Luljanus synagris and Ocyurus chrysurus were hatchery-raised specimens from parentscaptured in Miami

Tissues (eye heart liver and muscle) were dissected and stored at -80OC until use The tissues(02-03 g) were minced homogenized in ice-cold distilled water (025 ml) and centrifuged at 13000x g for 10 to 20 sec at room temperature Using the tissue extracts horizontal starch gel electrophoresiswas performed for 10 to 14 h at 75-100 volts at 4degC Electrophoresis buffers and tissues for 15 enzymesystems used are listed in Table 2 The staining procedures were those described by Murphy et al(1990) Using allele frequency data Neis genetic distance (D) (Nei 1978) was calculated Based onthe D values unweighted pair group method using arithmetic averages (UPGMA) (Sneath and Sokal1973) for cluster analysis and neighbor-joining method (NJM) (Saitou and Nei 1987) for additivetree analysis were performed to infer phylogenetic relationships among species

For morphometric analysis 12 characters of the skull were measured to the nearest 01 mm (FigI) Since the skulls of three out of four specimens of L vivanus were damaged this species was notincluded for morphometric comparison Except for frontal (FW) and parietal (PW) widths all char-acters have distinct apex segment or projections to be measured Transverse distance between dorsa]apices of eye-orbits were measured as the frontal width (FW) and that between parietal-frontal sutureswas adopted as parietal width (PW) Ratios of the eleven characters were calculated for the standardskull length (SSL) which was the distance from the tip of prevomer to ventral border between skulland atlas Discriminant analysis (DIA) was performed using ratios of the eleven characters in orderto calculate Mahalanobis distance between species One-way analysis of variance (ANOY A) wasapplied to the arc-sign transformed ratios and Tukcys test was performed for ranking species in eachcharacter

RESULTS

Electrophoresis -Interpretations for enzyme-encoding presumptive gene loci wererelatively simple in most enzyme systems except for calcium binding protein(Cbp) and malate dehydrogenase (Mdh) Although multiple bands or zones wereobserved in the gels stained for calcium binding protein only the most anodalzone was scored reliably in comparison between species Two or four bandingpatterns were observed in Mdh staining for muscle extract (Fig 2) In all speciesthe most cathodal band was interpreted as a mitochondrial form of MDH poly-peptide because of the apparent lack of affinity with the anodal polypeptides InL anais (La) L vivanus (Lv) and R aurorubens (Ra) it was assumed that thethree most anodal bands consisted of two homodimers of Mdh-l and Mdh-2

Table I Seven snapper species used in this study

Subfamily

Lutjaninae

Genus

Luljanus

OcyurusRhombopliles

Species Common name Abbreviation

L analis mutton LaL apodus school master LapL griseus gray LgL synagris lane LsL vivanus silk LvO chrysurus yellow tail OcR aurorubens vermilion Ra

510 BULLETIN OF MARINE SCIENCE VOL SO NO3 1992

Table 2 Enzymes and electrophoresis buffers used in this study

Enzyme Locus Tissue- Buffert

Acid phosphatase Acp L TC6Alcohol dehydrogenase Adh L TVBAspartate aminotransferase Aat-J L TC6

Aat-2 LCalcium binding proteins Cbp M TVBGlycerol-3-phosphate dehydrogenase G3pdh-J L TC6

G3pdh-2 MGlucosc-6-phosphate isomerase Gpi-J ML TC6

Gpi-2 HL-Idotol dehydrogenase Iddh L TC6Isocitrate dehydrogenase Idh-I HM TC6

Idh-2 LLactate dehydrogenase Ldh-A EHM TC6

Ldh-B EHLLdh-C E

Malate dehydrogenase Mdh- HM TC6Mdh-2 HMmMdh HM

Malic enzyme Me- EHM TC6Me-2 EHM

Mannose-6-phosphate isomerase Mpi HM TC6Phosphoglucomutase Pgm EHM TC6Phosphogluconate dehydrogenase Pgdh LM TC6Pyruvate kinase Pk HL TC6Superoxide dismutase Sod L TC6bull E eye H heart L liver M musclet TC6 (Tris-citrate pH 60) and TVB (Tris-Verscne-Borate pH 80) from Selander et al (1971)

subunits and the heterodimer between them In L vivanus a homodimer of Mdh-2subunits was assumed to have almost the same electrophoretic mobility withthat of the mitochondrial form On the other hand in L apodus (Lap) L griseus(Lg) L synagris (Ls) and O chrysurus (Oc) only one anodal band was observedWe tentatively assumed that Mdh-l and Mdh-2 subunits had almost the sameelectrophoretic mobility in these four species

Aat-2 G3pdh-2 Jdh-J Ldh-A Ldh-C and Sod were fixed for one allele for allspecies examined Allele frequencies of the other nineteen loci are shown in Table3 Neis genetic distances (0) between species are calculated from data at all 25loci (Table 4) The 0 values ranged from 0178 to 0898 with a mean of 0566plusmn 0207 between five congeneric species of Lutjanus from 0585 to 0975 witha mean of 0687 plusmn 0148 between five species of Lutjanus and O chrysurus from0603 to ] 177 with a mean of 0869 plusmn 0224 between five species of Lutjanusand R aurorubens and 0877 between O chrysurus and R aurorubens The intra-(between five species of Lutjanus) and inter- (between species of Lutjanus and Raurorubens) generic averages of the genetic distances were significantly different(one-way ANOVA F = 6245 P lt 005) suggesting that the electrophoreticcomparison supports taxonomic separation between the two genera On the otherhand the averaged genetic distance between species of Lutjanus and O chrysuruswas not different with that between five species of Lutjanus (one-way ANaYAF = 1243 P gt 01) Cluster (UPGMA) and additive tree (NJM) analyses givesimilar impressions for the species relationships (Fig 3 A B) UPGMA suggeststhat there could be at least two groups within Lutjanus where L apodus (Lap)and L griseus (Lg) (gray snapper group) are clustered separately from the othercluster containing L analis (La) and L vivanus (Lv) (red snapper group) and L


SSL

Figure I Lateral (A) dorsal (B) and ventral (C) views of the skull of Lutjanus griseus showing the12 characters measured Abbreviations are (EPI) inner epiotic width (EP2) outer epiotic width(ETW) ethmoid width (EVD) eye-vomer distance (EXW) exoccipital width (FW) frontal width(LEW) lateral ethmoid width (PVW) prevomer width (PW) parietal width (SH) skull height (SPW)sphenotic width and (SSL) standard skull length

512 BULLETIN OF MARINE SCIENCE VOL 50 NO3 1992

+

----====---- -----------~_-----_ ----_-4MJh-l

- MIh-2~-- rrMJh

0---------------------Lg Ls Lv Ra La Oc Lap Lv

Figure 2 Electrophoretic phenotypes of malate dehydrogenase (MDH) in muscle of seven snapperspecies In each species the most cathodal band was interpreted as a mitochondrial form (mMdh)One of L apodus (Lap) is heterozygote in mMdh locus The three most anodal bands in L anais(La) L vivanus (Lv) and R aurorubens (Ra) consist of two homodimers of Mdh-l and Mdh-2 subunitsand the heterodimer between them Homodimer of Mdh-2 subunits in L vivanus shows the samemobility with that of mMdh Mdh-l and Mdh-2 homodimers and their heterodimers were assumedto have the same electrophoretic mobility in L apodus (Lap) L griseus (Lg) L synagris (Ls) and Ochrysurus (Oc)

synagris (Ls) Similarly NJM indicates that the gray snapper group (Lap and Lg)is located distantly from the red snapper group (La and Lv) and L synagris (Ls)is located in between the two groups On the other hand the relationships amongspecies of Lutjanus and the two outgroup species O chrysurus and R aurorubensmay be better illustrated or understood by NJM than by UPGMA in which Ochrysurus keeps medium and similar distance from the gray snapper group (Lapand Lg) and from L analis (La) and L synagris (Ls) and R aurorubens is moredistant from the gray snapper group and O chrysurus but maintains mediumdistance from the red snapper group (La and Lv)

Morphometry -Ratios (expressed as percent) of the II skull characters wereplotted against standard skull length (SSL) (Fig 4) Since a relatively wide andsimilar range of body sizes (juvenile through adult) was examined in L griseus(Lg) L synagris (Ls) and O chrysurus (Oc) ratios which change through devel-opment (ie allometry) may be postulated In all eleven characters significantlypositive or negative relationships between the ratios and standard skull length(SSL) were observed at least in one of the three species and those species plotsare indicated with asterisk () in the figure An increasing tendency was observedin ratios of the ethmoid width (ETW) and eye-vomer distance (EVD) of all threespecies in those of the exoccipital (EXW) lateral ethmoid (LEW) and sphenotic(SPW) widths ofO chrysurus in that of the frontal width (FW) of L grise us andO chrysurus and in those of the prevomer width (PVW) and skull height (SH)


Table 3 Allele frequencies at 25 enzyme loci of seven snapper species

Species La Lap Lg Ls Lv Oc RaLoci Alleles 7 7 19 14 4 22 5

Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000

Average number of individuals examined per locus Loci fixed in one allele for all species are Aal-2 G3pdh-2 Idh-I Ldh-A Ldh-Cand Sod


Table 4 Genetic distances (Nei 1978) among seven snapper species

La Lap Lg Ls Lv Oc Ra

LaLap 0725 0566 plusmn 0207Lg 0782 0178 0687 plusmn 0148 0869 plusmn 0224Ls 0441 0438 0543Lv 0428 0753 0898 0470Oc 0606 0679 0584 0590 0975Ra 0603 1036 1177 0897 0631 0877

of L synagris and O chrysurus A decreasing tendency was observed in ratios ofthe inner and outer epiotic (EP I and EP2) and sphenoid (SPW) widths of Lsynagris and in that of the parietal width (PW) of L griseus and L synagris Weassumed that the ratios showing positive or negative relationships with the stan-dard skull length (SSL) would become constant after certain threshold(s) In factthe plotted curves of most ofthe characters tend to be horizontal as size increasesTherefore it is not correct to use all of the individual measurements To lose aslittle data as possible we defined the threshold at a standard skull length (SSL)of about 40 mm although naturally the threshold if any might be differentbetween species or characters Specimens whose SSL was larger than this thresholdwere selected and the ratios were used for ANOV A and DIA Averages with thestandard deviation of the ratios of eleven characters and the results of ANOV Awith Tukeys test are shown in Table 5

All ratios but one (ETW) showed heterogeneity among species The cranialpeculiarity of R aurorubens (Ra) is quite evident in which the ratios of EP IEP2 EXW FW LEW PW and SPW are predominantly larger whereas those ofEVD and PVW are smaller than in Lutjanus This indicates that the skull of Raurorubens is proportionally very wide compared with those seen in Lutjanusexcept for the prevo mer widths (PVW) The skull of O chrysurus has proportionalcharacters similar to those of R aurorubens in which ratios of frontal (FW) andparietal (PW) widths are larger whereas that of prevomer width (PVW) is smallerthan in Lutjanus All species of Lutjanus had similar skull morphometry exceptthat L analis had larger ratios in eye-vomer distance (EVD) and skull height (SH)

A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus

R aurorubensFigure 3 Cluster analysis (A) by UPGMA and additive tree analysis (8) by NJM using Neis geneticdistance values among seven snapper species See Table I for abbreviations


c

0 90 ~

S~ OJbulllt0 0

0Cl

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bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~

0 0 0 II J ~ c~ q M MlSSHS 1SSIMdS 0

~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~

~~ ~~I) I) ~ tlbull0 otbull tlcon l

c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl

N CllSS1M31 1SSfMltd 1SSlMdtl

0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

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~ Cl

tlOJ

~ 0 0 0 ~ ~ ~ ~ ~ ~ ~q M N ~ q M 155(013 1SSMX3 1SSMl 0-0 gtlt~ ~

bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N

oo~middot 1SSHd3 1SSIld3 1SSIML3 -iOJbulllOIl

LZ


Table 5 Averages (plusmnSD) and the range of the standard skull length (SSL) above hypothetical threshold(ca 40 mm) and averages of ratios (plusmnSD) of I I characters in six snapper species

Species La Lap LgCharacters 4 3 6

SSLt (mm) 802 plusmn 118 634 plusmn 29 584 plusmn 97(range) (673-974) (595-663) (392-708)

PEl OI91 plusmn 0003bsect 0171 plusmn 0003 0 I62 plusmn 0009ltEP2 0301 plusmn 0008middotb 0290 plusmn 0003middotb 0287 plusmn OOIObETW 0225 plusmn 0004middot 0220 plusmn 0009middot 0218 plusmn 0004EVD 0380 plusmn 0012 0336 plusmn 0007b 0305 plusmn 0010EXW 0422 plusmn 0007bc 0396 plusmn 0004lt 0419 plusmn 0003FW 0243 plusmn 0015b 0218 plusmn 0012b 0217 plusmn 0004bLEW 0263 plusmn 0004b 0238 plusmn 0008b 0253 plusmn 0006bPVW 0157 plusmn 0010 015 I plusmn 0002b 0154 plusmn 0004bPW OI39 plusmn 00 4lt 0155 plusmn 0004 0142 plusmn 0012ltSH 0517 plusmn 0004 0454 plusmn 0004lt 0451 plusmn 0006ltSPW 0435 plusmn 0008lt 0427 plusmn 0007lt 0441 plusmn 0012ltbull Number of individuals examinedt See Figure 2 for the abbreviationssect Averages carrying different superscripts are significantly different from one another

than the other species Differences between genera may be roughly estimated bycomparing the counts of significantly different characters (see Ta-lle 6 abovediagonal) where those between congeneric species within Lutjanus ranged fromo to 3 those between species of Lutjanus and O chrysurus ranged from 3 to 6and those between species of Lutjanus and R aurorubens ranged from 5 to 9Although Mahalanobis distances (Table 6 below diagonal) were significantly cor-related with the counts of significantly different characters (r = 0838 P lt 0005)a few discrepancies between these values are observed For example O chrysurusshows moderate counts of significantly different characters from Lutjanus butthe Mahalanobis distance are comparable with those of L analis This indicatesthat Ocyurus has a number of significantly but slightly different characters fromspecies of Lutjanus while L analis has fewer significantly and eminently differentcharacters from its congeneric members Thus Mahalanobis distance may bemore appropriate to illustrate morphometric difference among species along withthe genetic differentiation Cluster analysis (UPGMA) of Mahalanobis distances(Fig 5) indicates that L synagris (Ls) is morphometrically more similar to graysnappers (Lap and Lg) than to red snappers (La) and that unlike R aurorubensO chrysurus is not clustered out of Lutjanus

DISCUSSION

The external characters by which Rivas (1966) strictly separated three groupsin the genus Lutjanus are listed in Table 7 Lane and red snapper groups share

Table 6 Number of characters which are significantly different (by Tukeys test) (above diagonal)and Mahalanobis distance (by DIA) (below diagonal) between species

Species La Lap Lg La Oc Ra

La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p

474 plusmn 22 529 plusmn 64 473 plusmn 16(447-497) (402-584) (454-492)

0177 plusmn 0013bc 0184 plusmn 0005b 0214 plusmn 0004 1992 lt00010277 plusmn 0009b 0298 plusmn 00120b 0323 plusmn 0006 830 lt00010217 plusmn 0004 0215 plusmn 0008 0209 plusmn 0003 234 gt0050306 plusmn 0005bc 0304 plusmn 0007c 0253 plusmn 0004d 6852 lt00010441 plusmn OOIOb 0457 plusmn 0014b 0551 plusmn 00050 9369 lt00010198 plusmn 0007b 0336 plusmn 0016 0363 plusmn 0015 10470 lt00010256 plusmn 0005b 0260 plusmn 0008b 0314 plusmn 0009 365 lt00010161 plusmn 0004 0134 plusmn 0003c 0144 plusmn OOOlbc 4469 lt00010129 plusmn 0005lt 0193 plusmn 0015b 0223 plusmn 0009 3058 lt00010480 plusmn 0007bc 0500 plusmn 0017b 0474 plusmn OOOlbc 2519 lt00010463 plusmn OOl2bc 0489 plusmn 0014b 0556 plusmn 0010 5883 lt0001

six out of the seven characters coinciding with cluster analysis based on the geneticdistance in the present study Conversely our skull morphometric analysis sup-ports Vergara (1980) who categorized gray and lane snappers as a sister group ofred snapper This discrepancy might be rectified by assuming the lane snappergroup to be more of an intermediate form than Vergara (I980) expected Forexample Rivas (1966) included L buccanella (blackfin snapper) in the red snappergroup whereas Vergara (1980) placed this species in the lane (or mahogony)snapper group As shown by an additive tree analysis lane snapper (Ls) is indeedsituated in a position intermediate between the gray and red snapper groupsTherefore rather than three distinct groups in this genus of the western Atlanticsnappers we suggest that there are two well-defined groups (gray and red) asalready suggested by Rivas (1966) and Vergara (1980) and one genetically andmorphologically plastic group (lane snapper group) which possesses affinity forthe two distinct groups

L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens

bull50Mahalanobis Distance

bullo

Figure 5 UPGMA cluster analysis using Mahalanobis distances based on II skull morphometriccharacters among six snapper species


Table 7 Characters Rivas (1966) used for grouping Luljanus species of the Western Atlantic

Group

Character Gray Lane Red

Scales above opercle 2-3 rows 4-7 4-7Lateral scales 40-48(41-47) 46-53(47-51) 46-53(47-51)Jaws subequal or upper lower is longer lower is longer

is longerAccessory lateral usually absent present present

lines on caudal finDorsal rays 14 12 14Lateral spot absent present present or absentColor not red usually red usually red

Species L apodus L mahogoni L analisL griseus L synagris L buccanellaL cyanopterus L campechanusL jocu L purpureus

L vivan

There has been little doubt that Rhombopliles should stand as a mono typicgenus and this is supported by both our electrophoretic and morphometric anal-yses Within the three genera Rhomboplites may be the earliest offshoot as alreadysuggested by Johnson (1981) As is well known R aurorubens and O chrysurusshare some characters such as pterygoid teeth high numbers of gill rakers a deeplyforked caudal fin and small canines Furthermore in the present study we ob-served proportionally wider frontal and parietal bones and narrower prevomerof these two species than in Lutjanus Therefore one might be tempted to considerR aurorubens and O chrysurus as sister groups distinct from Lutjanus Howeverour biochemical and morphometric analyses indicate a closer relationship betweenspecies of LUljanus and O chrysurus and could not even rule out inclusion of Ochrysurus into the genus Lutjanus Therefore the similar characters shared be-tween O chrysurus and R aurorubens could be convergence for ecological ad-aptation Thus the status of Ocyurus as a monotypic genus is open to challengeTo test this hypothesis biochemical comparisons between Atlantic and Pacificsnappers would be useful because separation of the two genera LUljanus andOcyurus must have occurred before closing of the Isthmus of Panama

ACKNOWLEDGMENTS

We thank C R Robins University of Miami for his kind reading and valuable comments on themanuscript and K A Kilpatrick for helping us to use the SYST AT program M E Clarke is alsogratefully acknowledged for providing us valuable snapper samples This research was supported byNOAACIMAS contract NA90-RA-H-00075

LITERATURE CITED

Allen G R 1985 FAO species catalogue Snapper of the world An annotated and illustratedcatalogue oflutjanid species known to date FAO Fish Synop No 125 Vol 6 208 pp

Anderson W D Jr 1986 Review of the systematics of the fishes of the family Lutjanidae (Per-ciformes Percoidei) the snappers Pages 1-31 in S Ralston and J 1 Polovina eds Biology andfisheries management of tropical groupers and snappers Westview Press Boulder Colorado

Evermann B W and M C Marsh 1900 The fishes of Porto Rico Bull US Fish Comm (1899)51-350

Johnson G D 1981 The limits and relationships of the Lutjanidae and associated families BullScripps Inst Oceanogr 24 1-114

Murphy R W J W Sites Jr D G Buth and C H Haufler 1990 Proteins I isozyme electro-

CHOWANDWALSHPHYLOGENETICSTUDYOFATLANTICSNAPPER 519

phoresis Pages 45-126 in D M Hillis and C Moritz eds Molecular systematics Sinauer AssocMassachusetts

Nci M 1978 Estimation of average heterozygosity and genetic distance from a small number ofindividuals Genetics 89 583-590

Rivas L R 1966 Review of the Lutjanus campechanus complex ofred snappers Q J FI AcadSci 29 117-136

Saitou N and Nei M 1987 The neighbor-joining method a new method for reconstructingphylogenetic trees Mol BioI Evol 4 406-425

Selander R K S Y Smith S Y Yang W E Johnson and J B Gentry 1971 Biochemicalpolymorphism and systematics in the genus Peromyscus I Variation in the old-field mouse(Peromyscus polionotus) Stud Genet VI Univ Texas Pub I 7103 49-90

Sneath P H and R R Sokal 1973 Numerical taxonomy W H Freeman San Francisco 573 ppVergara R R 1980 Phylogenetic considerations on the Cuban species of the genus Lutjanus

(Lutjanidae Perciformes Teleostei) Acad Cienc Cuba no 113 39 pp (In Spanish with Englishabstract)

DATEACCEPTED June 3 1991

ADDRESS Division of Marine Biology and Fisheries RSMAS University of Miami 4600 Rick-enbacker Causeway Miami Florida 33149-1098


between species ofthe different genera or between species within the genus Lutja-nus are still unclear

For biochemical systematic purposes we have collected five species in the genusLutjanus and two in the two monotypic genera Parallel analyses of enzymeelectrophoresis and skull morphometry were performed for these seven speciesIn this paper we report the results and discuss the phylogenetic relationshipsamong the three genera and the seven species

MATERIALS AND METHODS

Seven snapper species used in this study (Table I) were mostly caught in Miami or Key West areasA few of Luljanus synagris and Ocyurus chrysurus were hatchery-raised specimens from parentscaptured in Miami

Tissues (eye heart liver and muscle) were dissected and stored at -80OC until use The tissues(02-03 g) were minced homogenized in ice-cold distilled water (025 ml) and centrifuged at 13000x g for 10 to 20 sec at room temperature Using the tissue extracts horizontal starch gel electrophoresiswas performed for 10 to 14 h at 75-100 volts at 4degC Electrophoresis buffers and tissues for 15 enzymesystems used are listed in Table 2 The staining procedures were those described by Murphy et al(1990) Using allele frequency data Neis genetic distance (D) (Nei 1978) was calculated Based onthe D values unweighted pair group method using arithmetic averages (UPGMA) (Sneath and Sokal1973) for cluster analysis and neighbor-joining method (NJM) (Saitou and Nei 1987) for additivetree analysis were performed to infer phylogenetic relationships among species

For morphometric analysis 12 characters of the skull were measured to the nearest 01 mm (FigI) Since the skulls of three out of four specimens of L vivanus were damaged this species was notincluded for morphometric comparison Except for frontal (FW) and parietal (PW) widths all char-acters have distinct apex segment or projections to be measured Transverse distance between dorsa]apices of eye-orbits were measured as the frontal width (FW) and that between parietal-frontal sutureswas adopted as parietal width (PW) Ratios of the eleven characters were calculated for the standardskull length (SSL) which was the distance from the tip of prevomer to ventral border between skulland atlas Discriminant analysis (DIA) was performed using ratios of the eleven characters in orderto calculate Mahalanobis distance between species One-way analysis of variance (ANOY A) wasapplied to the arc-sign transformed ratios and Tukcys test was performed for ranking species in eachcharacter

RESULTS

Electrophoresis -Interpretations for enzyme-encoding presumptive gene loci wererelatively simple in most enzyme systems except for calcium binding protein(Cbp) and malate dehydrogenase (Mdh) Although multiple bands or zones wereobserved in the gels stained for calcium binding protein only the most anodalzone was scored reliably in comparison between species Two or four bandingpatterns were observed in Mdh staining for muscle extract (Fig 2) In all speciesthe most cathodal band was interpreted as a mitochondrial form of MDH poly-peptide because of the apparent lack of affinity with the anodal polypeptides InL anais (La) L vivanus (Lv) and R aurorubens (Ra) it was assumed that thethree most anodal bands consisted of two homodimers of Mdh-l and Mdh-2

Table I Seven snapper species used in this study

Subfamily

Lutjaninae

Genus

Luljanus

OcyurusRhombopliles

Species Common name Abbreviation

L analis mutton LaL apodus school master LapL griseus gray LgL synagris lane LsL vivanus silk LvO chrysurus yellow tail OcR aurorubens vermilion Ra









Ldh-B EHLLdh-C E







SSL



+

----====---- -----------~_-----_ ----_-4MJh-l

- MIh-2~-- rrMJh








Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000








A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED
























Ldh-B EHLLdh-C E







SSL



+

----====---- -----------~_-----_ ----_-4MJh-l

- MIh-2~-- rrMJh








Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000








A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED

















SSL



+

----====---- -----------~_-----_ ----_-4MJh-l

- MIh-2~-- rrMJh








Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000








A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED

















+

----====---- -----------~_-----_ ----_-4MJh-l

- MIh-2~-- rrMJh








Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000








A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED



















Acp 100 0000 1000 1000 1000 0000 0000 000060 1000 0000 0000 0000 1000 1000 1000

Adh 150 0000 0000 0000 0094 0000 0000 0000100 1000 1000 1000 0906 1000 1000 000090 0000 0000 0000 0000 0000 0000 1000

Aat- 100 1000 0000 0000 1000 1000 1000 000090 0000 0000 0000 0000 0000 0000 100085 0000 1000 1000 0000 0000 0000 0000

Cbp 140 0000 1000 1000 0000 1000 0000 0000130 1000 0000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 0000 1000 1000

G3pdh- 110 0000 0000 0000 0000 0000 1000 0000100 0000 1000 1000 1000 0000 0000 000080 1000 0000 0000 0000 1000 0000 1000

Gpi- 100 1000 1000 1000 1000 0000 0167 000090 0000 0000 0000 0000 0000 0750 100080 0000 0000 0000 0000 1000 0083 0000

Gpi-2 200 0000 1000 1000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 1000

Iddh 250 0000 0000 0000 0000 0000 1000 0000180 0000 1000 1000 0000 0000 0000 0000100 1000 0000 0000 1000 1000 0000 1000

Idh-2 125 0000 0000 0000 0031 0000 0000 0000100 1000 0000 0000 0969 1000 0000 083385 0000 1000 1000 0000 0000 1000 0167

Ldh-B 100 0000 1000 0000 1000 1000 0000 100095 0000 0000 0000 0000 0000 1000 000085 0000 0000 1000 0000 0000 0000 000060 1000 0000 0000 0000 0000 0000 0000

Mdh- 110 1000 0000 0000 0000 1000 0000 1000100 0000 1000 1000 1000 0000 1000 0000

Mdh-2 100 1000 1000 1000 1000 0000 1000 000060 0000 0000 0000 0000 1000 0000 1000

mMdh 100 1000 0900 1000 1000 1000 1000 000070 0000 0100 0000 0000 0000 0000 1000

Me-l 110 0000 0000 0000 0000 0000 0000 1000100 1000 1000 0000 1000 1000 0000 000090 0000 0000 1000 0000 0000 1000 0000

Me-2 105 0000 0000 0000 0000 0000 0000 1000100 1000 1000 1000 1000 0000 1000 000095 0000 0000 0000 0000 1000 0000 0000

Mpi 110 1000 1000 1000 0000 1000 0000 1000100 0000 0000 0000 1000 0000 1000 0000

Pgm 125 0000 1000 0000 0000 0000 0000 0000100 0000 0000 0000 1000 1000 0000 000095 0000 0000 1000 0000 0000 0000 000080 1000 0000 0000 0000 0000 1000 1000

Pgdh 115 0000 0000 0000 0000 0000 1000 0000100 1000 0000 0000 1000 1000 0000 000095 0000 0875 1000 0000 0000 0000 100085 0000 0125 0000 0000 0000 0000 0000

Pk 100 0000 1000 1000 1000 1000 0000 000095 1000 0000 0000 0000 0000 1000 1000








A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED






















A L apodus

L griseus

L analis

O chrysurus

R aurorubens

BL apodus



c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED

















c

0 90 ~

S~ OJbulllt0 0

0Cl

~ bulllt8

~~~I)

bilI)

LZ0 IIl

IIlVJ

~ lt

ot~ ~


~h~IHll(~ lVJ

HttH ~ltbullOIl

0c ~


c~ ltjM lt0 ~ ~ ~ ~ q N ~ ~ OIl


0 ~0

0~ C ~

lt~ ot

gt-on -

C~ III 0

III C0

(lJbull ~OJ0-

~ Cl

tlOJ


bull~~S~8amp ~ IIl

Uotbull

~ otltu

~ ~~1Il bullIII

0bull

-0on 9

~~ ~ 0 ~ g re 0 N N


LZ







DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED






















DISCUSSION




La 2 3 2 5 8Lap 130 0 I 6 7Lg 170 73 0 6 8Ls 188 122 88 3 9Oc 217 166 142 181 5Ra 479 401 350 357 290


Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED

















Table 5 Extended

Ls Oc R4 7 3 F p




L apodus

L griseus

L synagris

L anais

O chrysurus

R aurorubens


bullo




Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED


















Group






L vivan


ACKNOWLEDGMENTS


LITERATURE CITED


























Documents

;BH FB LJB K ?HJ IAQEH@>GB E:LBHGKABIK ;>LO>>G K>N>G KG:II ...fsf.fra.affrc.go.jp/chow/paper/Lutjanus.pdf · 6NGEUSQRJQSGTKT' (BagXeceXgTg\baf Ybe Xaml`X(XaVbW\aZ