This document analyzes the evolutionary relationships of four grouper species (Epinephelus) found in the Gulf of California through biochemical analysis. Electrophoretic analysis of tissue extracts revealed that E. panamensis is closely genetically related to E. labriformis, suggesting a recent divergence. While E. panamensis displays some anatomical differences, the results do not support recognizing it as a separate subgenus. The relationships identified provide insight into the biogeography and phylogeny of the species in the Gulf.

1. BiochemicalSystematicsandEcology,Vol 16, No. 1, pp. 79-87, 1988. 0305-1978/88 $3.00+ 0.00
Printedin GreatBritain. PergamonJournalsLtd.
Biochemical Systematic Analysis of Evolutionary Relationships of
Groupers from the Gulf of California
LUIS GERARDO LOPEZ LEMUS
Division de Investigacion Biologia Marina, Centro de Investigaciones Biologicas de Baja California Sur, AC, Apartado
Postal No. 128, Le Paz, Baja California Sur 23060, Mexico
KeyWord Index--Epinephelus; Epinephelinae; Serranidae; congeneric fish; groupers; biochemical systematics; evolutionary
relationships; Sea of Cortez; biogeography.
Abstract--Four species of groupers (Epinephelus), with overlapping distribution ranges occurring in the middle and lower
regions of the Gulf of California, were studied in terms of biochemical, genetic and functional enzymic characters. The
electrophoretic analysis of extracts of tissues of these four congeners revealed that the species Epinephelus (Cephalopholis)
panamen$isis closely related geneticallyto E. labriformis and so with the rest of the major branch Epinephelus,thus suggesting a
recent evolutionarydivergencefrom that branch. Although this species might find its subgeneric rank justified in accordance with
the observed differences of its anatomical characters in relation to the restof the species studied, an alternate scheme of the group
phylogeny is proposedand the recognition that Cephalopholisis not avalid genus or subgenus of this American species. Finally,
the apparent relationship between these results and the biogeography of the involved species is discussed.
Introduction
Since Markert and Moiler [1] recognized the
biological significance of enzymes (multiple
molecular forms of enzymes), these gene markers
have proven to be powerful probes for the study
of genetic and organismic evolution. Isozymes
encoded in different alleles possess considerable
power for testing hypothesis within the fields of
evolution and systematics [2]. Electrophoretic
analyses have 12roved useful in studies of the
relationships and genetic structures of natural
populations of marine fish [3, 4]. Such methods
are valuable in providing additional data which
may be used to test hypotheses based on
morphological criteria.
In his comprehensive work on the morphology,
relationships and distribution of American
groupers, Smith [5] recognized five subgenera
within the major genus Epinephelus. One of
these, Cephalopholis, was considered only
because it has nine dorsal spines and lesser skull
modifications, not very important in the cladistic
sense. To test Smith's interpretations regarding
the phylogeny and systematics of Epinephelus
and intragenic differentiation in Cephalopholis,
(Received 7 February 1987)
79
an electrophoretic investigation of isozymes as
gene markers was conducted within four species
of the genus. A report of the relationships among
these four congeners forms the basis of this
study, including some notes on their biogeog-
raphy.
Results
Species analysed and collection sites
The species analysed are shown in Figs 1-4 and
the collection sites in Figs 5 and 6. The speci-
mens were collected mostly by snorkeling and
spear-gun in the western rocky shores of the
lower Gulf of California, including the southern-
most portion of the middle region (regions are
named according to Thomson and Gilligan [6]).
All specimens of E. acanthistius were bought
directly from local fishermen.
Morphometric and meristic data
In the present study, little emphasis has been
given to these data. The counts utilized were
designed only to establish the normal ranges of
variation among species. Not enough material
was obtained to justify an exhaustive study on
the geographic variation in these terms. The
counting of fin rays, however, is useful as a

2. 80 LUIS GERARDOLOPEZLEMUS
Epmephelus [CophalophoLis ) ponomensls ( Stelndachner, 1876)
Ep/nephe(us (Ep/nepheLu~ ) /obr/form/s (Jenyns , 1843]
t
Ep/nephelus (Ep/nephe(us ) oconth/st/us (Gilbert , 1892)
FIGS 1~,. SPECIESCONSIDEREDFORTHE PRESENTSTUDY.
Epmephelus (Epmephe(us) ono(ogus (G~LL,1864)
% ,,o.13o
Las Aromas
Tsland
(3"
~ef
~ San Francisquito 0
Island
Nkxxxxxxx US A. ~o~
I10"~30'
N
o
FIG.S.GEOGRAPHICLOCATIONOFCOLLECTIONSITES(SOUTHERNMOSTPORTIONOFTHEMIDDLESEAOFCORTEZ[6, Thomson,D.A.,
personalcommunication].
Partida Island I
~ Espirltu Santo ~
Island

3. ,FISH SYSTEMATICS AND RIOGEOGRAPHY 81
,0,%-];.-- I
-""~:~_ / Los ~ ® ~ I
Et So.geoto o. N I
24" ."---"--'-""~Cuevo de Leon 0o. 24- I
' /E°.no°° X
~ . ~ Los Borrites
t~e~ivera
, "o P~Lmo . I
.... % Los
XW'" h Son Jose /
o "~¢Yo " - / oo'
FIG. 6. GEOGRAPHIC LOCATION OF COLLECTION SITES (WESTERN COAST OF THE LOWERSEA OF CORTEZ[6, Thomson, D. A., personal
communication].
taxonomic character and was always recorded
(Tables 1 and 2). Morphometric data in groupers
were more satisfactory than meristics since most
of the specimens were big enough to obtain
considerable accuracy in the measurements.
Also, the species analysed have distinctive
characters shown by the measurements taken.
Table 3 shows the average values of these
measurements obtained from 10 specimens of
each species collected at the various study sites.
Electrophoretic analysis and genetic distances
The results of the electrophoretic analyses on the
enzyme loci examined (Table 4) are presented in
Table 5. Values of genetic distance [7], calcu-
TABLE 1. GENERIC CHARACTERS OF EPINEPHELUS AND
CEPHALOPHOLIS
Epinephelus Cephalopho/is
Head length long long
Caudal fin round or square round or square
Supramaxillary bone present present
Frontoparietal convergent convergent
skull crests anteriorly anteriorly
Supraethmoid wall present or absent absent
Suborbital width wide wide
Postocular process present present
Antrorse spine at absent absent
angle of preopercle
Dorsal rays IX-XI, 14-18 IX, 13-16
Anal rays III, 7-10 III, 8-9
Total pectoral rays 32-40 32-38
Transverse scale rows 80-129 54-69
Total gill rakers 21-29 16-27

4. 82 LUIS GERARDO LOPEZ LEMUS
TABLE 2 COMPARATIVE FEATURES OF THE FOUR SPECIES OF GROUPERS FROM THE SEA OF CORTEZ, GENUS EPINEPHELUS
CONSIDERED IN THE PRESENT STUDY
E panamensis E labriformis E analogus E acanthistius
Dorsal rays IX, 14 X1,16 17 X, 17 18 IX, 17
Anal rays 8 9 8 9
Pectoral rays 17 18 35 39 3940 33 37
Gill rakers 16 19 23 26 26 28 25 27
Caudal Peduncle saddle absent small, distinct on top absent absem
Longest dorsal spine 3 5 3 5 3 5 3 5
Caudal fin shape round round round or square round
Colour pattern green brown dark cross red-brown irregular white reddish-brown, red spots plain red
bands on the body spots on the body of even size
TABLE 3. AVERAGE VALUES OF PROPORTIONAL MEASURE-
MENTS OBTAINED FROM 10 RANDOMLY CHOSEN SPECIMENS
OF FOUR SPECIES OF GROUPERS FROM THE SEA OF CORTEZ"
Speciest
Measure Epan Elab Eana Eaca
Standard length (mm) 236.4 299.0 265.2 5863
Head length 4041 4257 4079 417.8
Head width 1648 192.0 1879 183.0
Head depth 268.8 257.2 252.0 316.8
Snout length 953 1027 91 4 972
Suborbital width 389 42 4 31 6 31 2
Interorbital width 53 3 64.2 68.1 694
Length of: orbital 749 75.8 82.4 93.3
Postorbital-head 238,9 258.3 2485 239 1
Maxillary 199.3 189.7 1824 2051
Lower jaw 207.8 1933 1863 2027
Snout-preopercle 289.7 288.6 286 2 2959
Supramaxillary length 646 575 56.2 75.4
Maxillary width 450 44 8 429 51.2
Body width 154.4 177.3 1667 163.9
Body depth 3581 343,5 343.8 4064
Caudalpeduncielength 1370 1108 120.1 128.0
Snout~lorsal fin 399 0 389.0 3735 393.9
Snout-pectoral fin 3801 370.9 357.9 3998
Snout-pelvic fin 4121 4234 3900 424.8
Length of: dorsal fin 529.9 5881 5967 5544
Depressed dorsal fin 6458 6659 681 3 675.5
Anal fin 1732 1672 177.5 196.6
Depressed anal fin 3186 2824 3106 358.5
Dorsalfinend~audal 141 5 118.3 1148 1233
Length of: caudal fin 185,4 186.9 2008 1848
Pectoral fin 252.8 2133 2352 3090
Pelvic fin 1833 166.7 1943 2177
Dorsal fin spine I 58.1 553 662 61.0
Dorsal fin spinelll 122.3 131.9 1401 158.0
Last dorsal fin spine 1150 107.6 1205 1641
Anal fin spine I 687 60.5 61 2 621
Anal fin spine ll 138.3 1105 1155 1271
Anal fin spinelll 1295 1153 120.8 146.6
Caudal fin base-upp, rays 222.4 216.0 2394 273.9
Caudal fin base-mid rays 244.5 214.3 250 1 294.7
Caudal fin base-low, rays 229.5 217.5 2320 270.6
"All measures are expressed in thousands of the standard length
t (Epan -Epinephelus panamensis; Elab - E. lebriformis; Eana -E, analo -
gus; Eaca-E. acanthistius).
lated from electrophoretic data, are given in
Table 6. The dendrogram of genetic distance
among the four species studied is shown in
Fig, 7.
TABLE 4. ENZYME LOCI SUCCESFULLYSCREENED FOR THE FOUR
SPECIES OF EPINEPHELUS CONSI DERED IN TH EPRESENTSTUDY
Protein E.C. Number Loci Structure Buffer*
Oxidoreductases
Lactate dehydro-
genase 1.1.127 1 4 Tetramer TC. EBT
Malate dehydro-
genase 1 1,1.37 1 3 Dimer TC, EBT
Malic enzyme 11.1.40 1 2 Tetramer TEMM
Superoxidedismutase1.151 1 1 2 Dimer TC, TEMM
Glucose dehydro-
genase 1.11,47 1 Dimer SP
Alcohol dehydro-
genase 11.11 1 2 D~mer EBT
Transferases
Phosphoglucomutase 27.51 1 2 Monomer TEMM, TC
Isomerases
Manose-P-isomerase 5.31 8 t Monomer EBT
Glucose-P-isomerase 5.3.1.9 1 2 Dimer EBT, TC
"All buffer systemsand stains have been previously described ~22-24,
27].
Discussion
Proportional measurements
If available, 10 specimens of each species were
measured in order to establish their corporal
proportions. The measurements were trans-
formed to thousands of the standard length and
are presented in Table 3. As the purpose of this
study was not to investigate the geographical
variation, the specimens were selected for
measurements which were not related to their
origin within the study area, The 37 measure-
ments taken were made to gather morphometric
data useful to the anatomical characterization of
the species. These data agree absolutely with
those obtained by Smith [5] in his comprehen-
sive work on American groupers. In order to make
the comparison among species easier, the
measurements given are average values obtained
from different specimens.

5. FISH SYSTEMATICS AND BIOGEOGRAPHY 83
TABLE 5. ALLELE FREQUENCIES OF POLIMORPHIC LOCI IN AND
AMONG FOUR SPECIES OF GROUPERS FROM THE SEA OF
CORTEZ (those loci not listed here were found fixed for the same allele
in the four species. Those values located below species names, show
sample size)
Species
Eana Epan Eaca E/ab
Locus* Allele (15) (25) (15) (25)
Ldh-a a -- -- 1.00 --
b -- 1.00 -- --
c 1.00 -- -- 1.00
m-Mdh a -- -- 1.00 --
b -- 1.00 -- 1.00
c 1.00 -- -- --
s-Mdh a -- -- 1.00 --
b 1.00 1.00 -- 1.00
Me a 1.00 1.00 -- 1.00
b -- -- 1.00 --
Mpi-a a 1.00 1.00 -- 1.00
b -- -- 1.00 --
Pgm-b a 1.00 1.00 -- 1.00
b -- -- 1.00 --
Gpi-a a -- -- 0.20 --
b 0.90 1.00 0.80 0.84
c 0.10 -- -- 0.16
Gpi-b a -- -- 1.00 --
b 1.00 -- -- --
c 1.00 -- -- --
Adh a -- 1.00 -- 1.00
b 1.00 -- 1.00 --
Sod-a a 1.00 1.00 -- 1.00
b -- -- 1.00 --
Sod-b a -- -- 1.00 --
b 1.00 1.00 -- 1.00
GId a 1.00 1.00 -- 1.00
b -- -- 1.00 --
"Names (abbreviations) of the investigated loci: lactate dehydroge-
nase (Ldh-a, Ldh-b, Ldh-c), malate dehydrogenase (m-Mdh-a, m-
Mdh-b, s-Mdh-b), malic enzyme (Me), manose phosphate isomerase
(Mpi-a), phosphoglucomutase (Pgm-a, Pgm-b), phosphoglucose
isomerase (Gpi-a, Gpi-b), alcohol dehydrogenese (Adh), superoxide
dismutase (Sod-a, Sod-b), glucose dehydrogenase (GId).
t(Eana-Epinephelus analogus; Epan-E. panamensis; Eaca-E. acan-
thistius; Elab-E. Labriformis). species abbreviations,
Some measurements were practically constant
for all species; others reveal trends within the
genus or resemble those within their own species
group as described by Smith [5]. The various
Epinephelus species showed remarkable differ-
ences in some characters (Table 3). For instance,
the maxillary length of E. acanthistius differs
notably from the rest of the studied species. In the
same way, the pelvic fin length is a well defined
character for this species and for its correspond-
ing species-group: such a measure is allometri-
cally negative.
E. panarnensis showed the third dorsal spine, a
short one, and the first anal spine is long in
comparison with those of the other species. The
taxonomical significance of these characteristics
will be discussed below.
Meristic characters
Some of these features were constant enough to
be of use in distinguishing genera and species
although, for this work, it was more important to
define which of these merisitic characters were
useful to identify interspecific relations. Never-
theless, as for all other taxonomic features,
meristic data varied independently of such rela-
tionships, so the loss of dorsal fin spines in E.
analogus (to 10) and E. acanthistius (to 9) will
be more readily understood when the data
includes all the characters for these and related
species, as it is being done by Randall and
Johnson (personal communication).
TABLE 6. GENETIC IDENTITY ESTIMATIONS IN FOUR SPECIES OF GROUPERS FROM THE SEA OF CORTEZ (GENUS EPINEPHELUS)
Species
E. panamensis E. labriformis E. analogus E. acanthistius
E. panamensis -- 0.8269 0.6941 0.4584
E. labriformis 0.190 -- 0.7393 0.5299
(3.42)
E. analogus 0.365 0.302 -- 0.5460
(6.57) (5.43)
E. acanthistius 0.780 0.635 0.605 --
(14.04) (11.43) (10.89)
I Over the diagonal, and of genetic distance, D= -In / below the diagonal [25].
The estimations are based on 16 apparent common loci to the four species. Inside brackets, below genetic distance values, approximated times of
divergence (in million years) based on Sarich's correlation [18] of genetic distance values and divergence times for vertebrates, are shown. These
correlation values were modified since Carlson et al. [17].

6. 84 LUIS GERARDO LOPEZ LEMUS
Genebc distance (D)
060 050 040 050 0 20 0 IO
I I
E ponomens/s
E (obr/forrn/s
EpmepheLus E omc]togus
E ooonth/st/us
1080 900 720 540 360 180
Divergence time (MILLion years)
FIG. 7. DENDROG RAM [26] OF GENETIC DISTANCE VALUES [7, 25]. Based on all possible pair comparisons of four species of groupers from the
Sea of Cortez, genus Epinephelus
Electrophoretic data and the systematics of
Epinephelus including notes on their
biogeograph y
The results obtained from the electrophoretic
studies shows that the two grouper species
mostly present in subtropical waters (E. pana-
mensis and E. labriformis) are more closely
related to each other than to the most tropical
species, E. acanthistius. The genetic distances
between pair combinations of the group E.
analogus-E, panamensis-E, labriformis are of
one half in relation to the distances between any
member of that group and E. acanthistius. The
genetic distances calculated from the electro-
phoretic survey of these four species of groupers
from the Sea of Cortez, can be discussed in
relation to the affinities based on morphological
characters. E. acanthistius differs from the the rest
of the species because of its maxillary length,
long pelvical fin rays and body size. The counts
on the dorsal fin rays are high in comparison with
its species group; it has 17 but only nine spines. If
the loss in the number of spines occured at the
posterior edge of the spined dorsal fin portion,
then this fact could have been accompanied by
the addition, at the same time, of two soft rays.
This fact is observed in E. analogus which
possesses one dorsal spine less in relation to the
rest of its congeners of the species-group con-
taining them.
It should be noted that E. acanthistius was
referred to the genus Cephalopholis for a long
time ([8] noted in [5] ) because of the possession
of nine dorsal spines. Nevertheless, considering
the rest of its characteristics, it has been clearly
assigned to the species-group E. niveatus within
the genus Epinephelus because it shares long
pelvic fin rays, deep body, red-chocolate color-
ation and dark pelvic fins [5].
There is a still much discussion on the differ-
ences and affinities among groupers. They have
been treated in extensive revisions [5, 9-1 5], in
all of which differences are found in relation to
treatment of levels of species or genera. While
some authors recognize genera, others perceive
them as subgenera, as in the case of Cephalo-
pholis [5, 14].
In the present work I have considered one
species of this genus, or subgenus of Epinephe-
lus according to Smith [5]. Cephalopholis differs
from the rest of Epinephelus in the possesion of
just nine dorsal fin spines and lesser skull
modifications. This genus contains a sole species
in the eastern Pacific, Epinephelus (Cephalopho-
lis) panamensis related genetically to Epinephe-
lus (Epinephelus) labriformis.
If the systematic position of E. acanthistius is
correct within Cephalopholis [8], it should be
expected that there would be a closer genetic
relation with E. panamensis and, as shown, that is
not the case (Fig. 7). Moreover, the genetic
affinities of E. panamensis are greater to E.
labriformis and E. analogus, subtropical and
temperate species, than to E. acanthistius.
To be useful in the determination of phyletic
lines, the characters used must be consistent in
and among the groups examined, and must be
subject to a minimum of individual variation. In
the groupers treated here, meristic data are useful
and the osteological features are very important.

7. FISH SYSTEMATICS AND BIOGEOGRAPHY 85
Some of the proportional measures serve to
separate major groups and to indicate also the
trends of these grou pars better than other charac-
ters. Coloration is the most useful character when
working in the field but it is of limited value in the
indication of phylogeny. The juvenile pattern, in
particular, is useful for the alignment of certain
species. Sometimes too much importance is
given to the general appearance of organisms,
but such subjective appreciations require support
from other sources so they can point out a more
accurate quantification of the observations.
The evolutionary scheme of the major branch
Epinephelus is far from being a complete one for
the species analysed with morphometric and
meristic characters. Smith [5] (Fig. 8a)identified
two distinct lineages for these species: a more
specialized trend of Epinephelus, and Cephalo-
pholis assubgenera (the observations and results
obtained are in complete agreement with those of
Smith [5]). Among them, E. labriformis the
affinities of which are difficult to establish, is
placed by Smith [5] close to the species-group E.
striatus because of structural similarities,
although he also suggests that this species might
be a recent immigrant form from the Indo-Pacific
region without close relatives in America. As a
definitive and more feasible solution to this
question, he proposed that E. labriformis is a
recent ramification of the clade Epinephelus, a
fact partially in accordance with the proposed
phylogenetic scheme of this work (Fig. 8b). The
species-group containing E. analogus also does
not possess close relatives in America and
probably evolved in the Indo-Pacific region too.
In contrast, the species-group containing E.
acanthistius and other species, probably evolved
in American waters. This fits in completely with
my proposed scheme in relation to the rest of the
species studied, apparently of non-American
origin according to Smith [5]. This biogeo-
graphic aspect will be discussed later.
From the previous results one might assume
that E. analogus and E. acanthistius evolved as
two different and independent lineages. In a
certain way this is in agreement with the scheme
of Fig. 8b. The dendrogram shows E. acanthistius
as a member of a line segregated independently
from the common stem Epinephelus approxi-
mately 15 million years ago, but E. analogus
comes from an ancestor with characteristics of E.
panarnensis- E. labriformis which subsequently
evolved to the actual form of E. analogus.
Another lineage which can be identified,
because of the secondary reduction of the
number of dorsal fin spines down to nine, gave
origin to the subgenus Cephalopholis including
the eastern Pacific species E. (C.) panamensis
studied here. This species showed great genetic
affinities with the rest of the groupers considered
A Epinephelus
r (Cepho(opholis)--E.ponorn~sis (I )
I
Il'r TIT"IV rEl oconth/sOus(TI)
I
L (Eplnephelus) . lobrlformls (111)
l anologus (1"v)
(EplnepheLus)~ I
~ CephaLophoUs--E.panornens/s
L__ TTr E. labrlformis
nTI2
T~r E.onologus
TT E. oconthistius
FIG. 8. COMPARATIVE DENDROGRAMS SHOWING THE POSSIBLE PHYLOGENETIC RELATIONS OF FOUR SPECIES OF GROUPERS FROM
THESEA OF CORTEZ. (A) Based on morpholgical characters [5]; (B) according to the genetic characters discussed in text. I, II, IV are the species-
groups. Subgenera appear inside parentheses.

8. 86 LUIS GERARDO LOPEZ LEMUS
thus suggesting that Cephalopholis is a recent
lineage derived from the same subgenus Epine-
phelus about 3.5 million years ago.
The electrophoretic survey of these species
species revealed the existence of enough fixed
allelic differences among them, a fact that assures
their status as separate species. However, the
kinship of E. panamensis with E. labriformis on
the basis of the relative mobilities of their proteins
and the analysis of their gene frequencies,
suggests a recent diversification from the major
genus Epinephelus.
On the basis of this work, I do not see any solid
argument (morphological, biochemical or gene-
tic) to recognize the name Cephalopholis as a
valid genus or subgenus at least for the American
specimens. Obviously, an integration of all avail-
able information on the systematics of these
species will be fruitful (as is being done by
Randall and Johnson, personal communication,
for the Indo-Pacific species) for there are still
some who recognize the species as a separate
group in accordance with small anatomical
differences.
The two species of temperate and subtropical
species, E. analogus and E. labriformis, and the
tropical E. panamensis show genetical affinities
but not anatomical ones along the coast of the
lower Gulf of California. Hubbs i16] pointed out
that such affinities are common in eastern Pacific
fish and are a result, probably, of equatorial
crossings in tropical compression periods during
the Pleistocene. Nevertheless, on applying the
genetic distance~ivergence time correlation of
ref. [17], modified from ref. [18] obtained from
other vertebrates, to the genetic distance separat-
ing these groupers a divergence time of 3.5 mil-
lion years is obtained for the most closely related
pair, E. panamensis-E, labriformis (Fig. 7). The
genetic distance value (D=0.190) separating
these species is also close to the average distance
between fish species-pairs separated by the
emergence of the Panama isthmus 1-19], an event
which occurred some three million years ago
[20]. The divergence times obtained from gene-
tic distance values (Fig. 7) show that the sepa-
ration of both clades occurred during the Plio-
cene; that the separation of E. analogus occurred
during the same time period (5-7 million years
b.p.) and that E. acanthistius has been separated
from all the others since the transition of the
Miocene-Pliocene (11-15 million years b.p.).
On the basis of the present phylogenetic
scheme, the most parsimonious reasoning on the
biogeography of the species examined has to be
done in terms of just proposing an open hypothe-
sis on which future research can be built. Since E.
panamensis and E.labriformis are the two species
which have most recently diverged from the
major stem of Epinephelus, there is still some
doubt as to whether they recently evolved in
American waters or if they have just arrived, in
geological time terms, from the Indo-Pacific
region. Both arguments could partially explain
the lack of close relatives in America.
The E. analogus case is different, because it is
clear that its species-group is completely of
Indo-Pacific origin. Within the proposed phylo-
geny this make some 'noise' if it is also assumed
that E. acanthistius and its species-group are of
American origin. If there is any way to fit the
scheme under the whole tropical eastern Pacific
Ocean region or the distribution ranges for these
species, E. analogus should be the earliest or the
oldest form of the analysed species complex in
the lower Gulf of California, because of its
confirmed Indo-Pacific origin among nearly all
American originated species, for two reasons.
(a) If it is accepted that E. labriformis and E.
panamensis are recently evolved species of
Epinephelus in America, and E.acanthistius is the
old American species, then E.analogusshould be
the earliest form because of its recent arrival to
American waters, or the oldest in terms of not
sharing common morphological or genetical
features in relation to the rest of the species.
(b) If it is accepted that both the former species
have recently evolved in the Indo-Pacific region,
the proposed scheme fits exactly with the
reasoning of the later arrival or divergence of E.
analogus and the antiquity of E. acanthistius as
the American originated species, as shown in the
proposed dendrogram of phylogenetic relation-
ships for these groupers (Figs 7, 8b).
Acceptance of the second hypothesis shows
the usefulness of electrophoretic data in the
elucidation of possible biogeographical relation-
ships, and not only systematic ones, of the
involved species.
Experimental
Meristic and morphometric data were recorded for all speci-

9. FISHSYSTEMATICSAND BIOGEOGRAPHY 87
mens collected. At the time of collection, sea surface
temperature, depth of collection, bottom type, individual's
activity and Secchi disk depth (a measure of water trans-
parency) were also recorded in order to obtain more informa-
tion about each collection and were used for other studies [3,
4, 21]. Eightyspecimens were examined, 15 of E. acanthistius
and 15 of E. analogus, 25 of E. labriformis and 25 of E.
panamensis.
Once all data were recorded, complete specimens or tissue
samples of eye, heart, liver and skeletal muscle were immedi-
ately frozen and transported to laboratoryfacilities where they
were preserved at -20 ° until biochemical-genetic processes
were carried out.
Tissuesample preparation, electrophoresis and histoctTemi-
cal stain. The methods for tissue sample preparation, electro-
phoresis protocols and interpretation of gel banding followed
those of ref. [22], and had been described elsewhere [4].
Each fish was screened for nine enzymes, identifying a total
of 16 gene-loci markers. These enzymes and their correspon-
dent loci are listed in Table 4, including the electrophoretic
buffer systems utilized for each. After the electrophoretic run,
the gel was sliced horizontally and each part stained for the
specific enzyme to be recorded according to the methods
described by refs [23, 24] modified according to availabilityof
materials and chemicals.
Calculations for allele frequencies, genetic distances and
dendrogram construction. The different electromorphs on gels
were interpreted in terms of modern molecular genetics
(allozymes), and allele frequencies were computed at the
various loci. The allele frequency data were then converted
into a measure of genetic distance among populations
following Nei's treatments [7, 25] correlating these coeffi-
cients with estimates of the approximate time of divergence
[17]. Finally, the genetic distance values were clustered in
dendrograms constructed using the UPGMA method of ref.
[26].
Acknowledgements--Thiswork was supported by a CIB-
Consejo Nacional de Ciencia y Tecnologia- Mexico (CON-
ACyT) grant PCECCNA/020963 and a CONACyT thesis-
scholarship No. 44182. I would like thank ProfessorsJoaquin
Arvizu and Francisco de Lachica for their helpful comments
upon earlier drafts of this paper. I am glad to acknowledge the
help of Mrs Maria Gomez and Ms Ana Luz AIvarez during the
preparation of the manuscript.
References
1. Markert, D. L. and Moiler, F. (1959) Proc. NatlAcad. ScL
U.S.A. 45, 753.
2. Wake, D. B. (1981) in Evolution Today, Proceedings of
the 2nd International Congress of Sysematic and Evolu-
tionary Biology (Scudder, G. G. E.and Reveal, J. L., eds)
pp. 257-270. Hunt Institute for Botanical Documen-
tation, Pittsburgh.
3. Lopez Lemus, L. G. (1985) CIBCASIO Trans. X.
4. Lopez Lemus, L. G. (1987) An. Esc. NaL Cienc. BioL IPN
(in press).
5. Smith, C. L. (1971) Bull. Amer. Mus. Nat. Hist. 146, 67.
6. Thomson, D. A. and Gilligan, M. (1983) in Island
Biogeography of the Sea of Cortez (Case,T. and Cody, M.
L., eds) pp. 98-129. University of California Press, Los
Angeles.
7. Nei, M. (1978) Genetics89, 583.
8. Meek, S. E.and Hildebrand, S. F. (1925) PubL FieldMus.
Nat. Hist. ZooL Set. 249, xv-xix+331.
9. Gosline, W. A. (1966) Proc. Calif, Acad. ScL 33, 91.
10. Johnson, G. D. (1983) Copeia 777.
11. Smith, C. L. (1961) FAOFish. BioL Synopsis23.
12. Smith, C. L. (1965) Am. Mus. Nat. Hist. Noviates 2207,1.
13. Johnson, G. D. and Keener, P. (1984) Bull. Mar. Sci. 34,
106.
14. Randall, J. E.and Ben-Tuvia, A. (1983) Bull. Mar. Sci. 33,
373.
15. Thomson, D. A., Findley, L. T. and Kerstitch, A. N. (1979)
ReefFishes of the Sea of Cortez: TheRocky-Shore Fishes
of the Gulf of California. John Wiley, New York.
16. Hubbs, C. L. (1952) Proc. 7 Pacific Sci. Cong. 3, 324.
17. Carlson, S. S., Wilson, A. C. and Maxson, R. D. (1978)
Science 200, 1183.
18. Sarich, V. M. (1977) Nature 266, 24.
19. Vawter, A. T., Rosenblatt, R. H. and Gorman, G, C. (1980)
Evolution 34, 705.
20. Keigwin, J. D., Jr (1978) Geology 6, 630.
21. Lopez Lemus, L. G. (1985) Mem. VIII Cong. NaL Zoo/.
Mex. 1,230.
22. Gorman, G. C., Soule, M., Yang, S.Y. and Nevo, E. (1975)
Evolution 29, 52.
23. Shaw, C. R. and Prasad, R. (1970) Biochem. Genet. 4,
297.
24. Harris, H. and Hopkinson, D. A. (1976) Handbook of
Enzyme Electrophoresis in Human Genetics. North Hol-
land Biomedical Press,Amsterdam.
25. Nei, M. (1972)Am. Nat. 106, 283.
26. Sokal, R. R. and Sneath, P. H. A. (1963) Principles of
Numerical Taxonomy. W. H. Freeman, San Francisco.
27. Selander, R. K., Smith, M. H., Yang, S. Y., Johnson, W. E.
and Gentry, J. B. (1971) Univ. TexasPubL 7103, 49.