PHYILOGENETIC RELATIONSHIPS ETWEEN MORPHS LOGICALLY SIMILAR ,tARBUS SPECIES, WITH REFERENCE TO TF MR TAXONOMY, DISTRII1 UTI N AND CONSERVATION. by JOHANNES SCHALK ENGEL RIECHT Thesis presented In fulfilment of the requirement For the degree PHILOSOPHY D '0) CTOR in ZOOLOGY in the FACULTY OF NATURAL SCIENCES at the RAND AFRIKAANS UNIVERSITY PROMOTOR: DR. F. H. VAN DER BANK CO-PROMOTOR: PROF. J. T. FERREIRA AUGUST 1996
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PHYILOGENETIC RELATIONSHIPS ETWEEN
MORPHS LOGICALLY SIMILAR ,tARBUS SPECIES,
WITH REFERENCE TO TF MR TAXONOMY,
DISTRII1 UTI N AND CONSERVATION.
by
JOHANNES SCHALK ENGEL RIECHT
Thesis presented
In fulfilment of the requirement
For the degree
PHILOSOPHY D '0) CTOR
in
ZOOLOGY
in the
FACULTY OF NATURAL SCIENCES
at the
RAND AFRIKAANS UNIVERSITY
PROMOTOR: DR. F. H. VAN DER BANK
CO-PROMOTOR: PROF. J. T. FERREIRA
AUGUST 1996
CONTENTS:
Table of contents Abstract Samevatting Acknowledgement Foreword
Chapter 1: Introduction - a rationale for the selection of goldie and
chubbyhead barb populations for phylogenetic studies. 1.1 - 1.8
Chapter 2: Morphological characteristics of the goldie and chubbyhead
barb populations studied. 2.1 - 2.10
Chapter 3: Isozyme and allozyme differences in four shortfin barb (Barbus
brevipinnis Jubb, 1966) populations with reference to an
undescribed Barbus species from the Transvaal, South
Africa. 3.1 - 3.16
Chapter 4: Allozyme differences between populations of chubbyhead barb
(Barbus anoplus Weber, 1897) and Marico barb (B. motebensis
Steindachner, 1894). 4.1 - 4.17
Chapter 5: Genetic relationships between Marico barb (Barbus
Figure 1: A graphic representation of points obtained by using SO CA
depicting the relationships between the populations
studied and the meristic characteristics measured. 2.6
Table 1: Sample size, species and localities where specimens of
chubbyhead and goldie barbs were collected for
morphometric and meristic measurements. Species listed
according to existing taxonomy. 2.7
Table 2: Morphological characteristics of goldie barb populations
studied. See Table 1 for locality details. 2.8
Table 3: Morphological characteristics of chubbyhead barb
populations studied. See Table 1 for locality details. 2.9
Table 4: Similarity (Jaccard, 1928) between the populations studied
based on meristic characteristics. See Table 1 for locality
details. 2.10
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
ODUCTDON
The taxonomy of freshwater fish in southern Africa is largely based on meristic
parameters (Gilchrist and Thompson, 1913; Barnard 1938, 1943; Groenewald,
1958; Crass, 1960, 1964; Jubb, 1966, 1967; Skelton, 1993). The parameters most
often used by these authors included counts of scales and fin rays. Morphometric
measurements have also been used to describe species of goldie barbs, e.g.
Jubb (1966) used proportional body measurements to compare B. brevipinnis with
B. viviparus. However, morphometric characteristics of populations may be
influenced by local environmental factors [i.e. the short fins of B. brevipinnis
(shortfin barb) are characteristic only of the Marite River population (Skelton,
1993)]. The purpose of this chapter is to evaluate if genetic studies would be
needed to supplement morphological characteristics to describe variations within
the goldie and chubbyhead barb groups of minnows.
MATE GALS D ETHODS
Morphological measurements and counts used in the present study included those
most often used in literature to describe the relevant species. For morphometric
comparisons within and between the goldie and chubbyhead barb populations and
species studied, the following measurements were taken from specimens: total
and standard length; head length and width; dorsal and anal fin length; orbital
diameter and gut length. Meristic measurements taken from fish specimens
included counts of lateral line and caudal peduncle scales, dorsal and anal fin rays
and the number of barbels.
2.1
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
Measurements (1/10mm) were taken with a vernier caliper (accuracy 0.02mm)
from 311 specimens representing 14 populations (Table 1). Because three of the
total of 17 populations included in the present study were frozen, no
measurements were taken from these to avoid unnecessary thawing of samples
before electrophoresis. Using existing research as a guideline (Jubb, 1966;
Skelton, 1988), body measurements were expressed as a percentage of standard
length for comparative purposes. SO MC i (Greenacre, 1990) was used to analyse
a contingency table of meristic count frequencies. SO CA projects two sets of
points (populations and meristic characteristics) into a multidimensional space,
after which it determines the position of each point in relation to a lower number
of dimensions. For further details refer to Greenacre (1987). Meristic counts were
transformed into a presence or absence matrix before it was statistically analysed,
using OS (Jaccard's (1928) similarity index).
RESULTS AND DISCUSS1 N
The results of the morphometric and meristic measurements for the goldie and
chubbyhead barbs studied are presented in Tables 2 and 3. These tables suggest
that the morphometric characteristics of the populations studied varied more within
than between populations and that the different species could not be differentiated
based on these values. Even in B. brevipinnis (shortfin barb) where short fins are
characteristic for the Marite River population, no clear separation between this
population and any of the other goldie or chubbyhead barb populations studied
was observed. During sampling, an effort was made to collect the largest
2.2
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
individuals available in the population. Based on these measurements, a size
difference exists between the goldie and chubbyhead barbs.
Several meristic differences were observed between the populations studied,
which could be of value to differentiate between the goldie and chubbyhead barbs
(Tables 2 and 3). In Fig. 1, SIIVICA differentiates between the goldie and
chubbyhead barbs on the X-axis (positive and negative respectively) based on the
following meristic characteristics:
The number of scales on the lateral line (11-12 and 13) corresponds with the
goldie barbs (G1-8) and (14 and 5) with the chubbyhead barbs (CH1-2 and
5; TU1-3).
The dorsal fin rays can be used to differentiate between the goldie barbs
(d2) and the chubbyhead barbs (dl).
The number of scales on the caudal peduncle is useful to differentiate
between the goldie (q1) and chubbyhead barbs (q2 and 3).
SIB; C differentiates between B. anoplus and the other chubbyhead barb
species on the Y-axis (negative and positive respectively) based on the following
meristic characteristics (Fig. 1):
Caudal peduncle scale counts (q3) correspond with B. anoplus (CH1-2
and 5) and (q2) with the other chubbyhead barb species (TU1-3).
The presence of two pairs of barbels (b2) in B. motebensis and B. gurneyi
(TU1-2 and 3) separates these populations from B. anoplus (CH1-2 and 5)
2.3
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
which has only one pair of barbels (b1).
Some characteristics did not corrrespond to any of the species studied (i.e. an
incomplete lateral line (Ii) was encountered only in B. anoplus from the Vaal River
(CH5) and B. motebensis from the Marico River (TU2)). The close grouping
between the populations within the goldie barb group (G1-8) in contrast to the
chubbyhead group (CH1-2 and 5; TU1-3) suggest that the species and
populations within the former group are meristically very similar. The similarity
index (Table 4) suggests two groups of goldie barbs consisting populations G1-4
and G8 (B. brevipinnis) and G5-7 (B. pallidus/neefi) respectively. These two
groups were 100% and 88% similar within and between the two groups
respectively. The latter suggests that G8 may be incorrectly classified as B.
pallidus. The chubbyhead group of barbs studied (CH1-2 and 5 ; TU1-3) were not
closely grouped in Fig. 1, which suggested meristic differentiation within and
between populations and species within this group. The similarity index shows OS
values ranging from 39% to 88% between the different populations and species
within the chubbyhead group of minnows and OS values from 7% to 33% between
the goldie and chubbyhead barb groups of minnows. This suggests that the
chubbyhead barbs show meristic differentiation, which cannot be explained in
terms of their present taxonomic status. It also shows that chubbyhead barbs can
easily be separated from the goldie barbs based on these meristic parameters.
Morphometric and meristic characteristics of populations can be powerful tools
2.4
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
to describe species and specie-strains, as suggested by Teugels (in press).
However, difficulties were experienced to differentiate between the species
because of the meristic differentiation within species of chubbyhead barbs and the
lack thereof between species of goldie barbs. Based on the characteristics
described above, it is evident that additional data is needed to clarify the
phylogenetic relationships of these fishes.
25
CH2 0
CH5 ❑
q2
b2 0
TU3 0
TU2 TU1 ❑
-al -di- 0 0
q1
d 11 2 Ic
0
11:2 3
14 b1
CH1 0
q3 0
li 15 9
Morpholoeical Characteristics of the Goldie and Chubbvhead Barb Populations Studied
LEGEND
11 = 26-27 lateral line scales al = AIII 5 anal fin rays 12 = 28-29 lateral line scales a2 = A1116 anal fin rays 13 = 30-31 lateral line scales li = incomplete lateral line 14 = 32-33 lateral line scales lc = complete lateral line 15 = 34-35 lateral line scales bl = 1 pair of barbels ql = 12 caudal peduncle scales b2 = 2 pairs of barbels q2 = 14 caudal peduncle scales G1-8 = goldie barbs q3 = 16 caudal peduncle scales CH1, 2 & 5 = B. anoplus dl = DIII 7 dorsal fin rays TU1-3 = other chubbyhead barbs d2 = DIII 8 dorsal fin rays
Figure 1 : A graphic representation of points obtained by using SINICA depicting
the relationships between the populations studied and the meristic
characteristics measured.
2.6
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
Table 1: Sample siz species nd Dc. ceDotB& s wf re speci ens of ch '-byh- -d and gs Idle barbs were c rDDE cted f r morsh.metrlc and meRistic measurrements. Species listed accordi g tw ex sting t xol a t.)my.
N Species Locality Latitude Longitude bbreviation
30 10 20
30 30 10
30 30 21 20
10 10 30 30
Chubbyhead barbs
B. anoplus Buffelskloofspruit A 24°47'S 30°30'E CH2 B. anoplus Ngagagane E 27°59'S 29°52'E CH1 B. anoplus Blesbokspruit C 26°11'S 28°23'E CH5
B. motebensis Ohrigstad River ° 24°53'S 30°36'E TU1 B. motebensis Kaaloog se Loop E 25°47'S 26°24'E TU2 B. gumeyi Msinduzi F 29°38'S 30°25'E TU3
Goldie barbs
B. brevipinnis Marite River' 24°47'S 31 °05'E G1 B. brevipinnis Sand River" 24°09'S 31 °02'E G2 B. brevipinnis Sterk River' 24°32'S 28°31'E G4 B. brevipinnis Grootspruie 24°29'S 27°51'E G3
B. pallidus Manzaan River K 27°38'S 30°53'E G8 B. pallidus Ngagagane E 27°59'S 29°52'E G7 B. pallidus Buffelskloofspruit A 24°47'S 30°30'E G6 B. neefi Ohrigstad River D 24°53'S 30°36'E G5
A
B
C G
E
F G
" I J
K
Tributary of the Crocodile River (Incomati River System). Tributary of the Buffels River (Tugela River System). Tributary of the Vaal River (Orange River System) Tributary of the Olifants River (Limpopo River System). Tributary of the Marico River (Limpopo River System). Tributary of the Mgeni River (Mgeni River System). Type Locality (Jubb, 1966), Tributary of Sabie River (Incomati River System). Tributary of Sabie River (Incomati River System). Tributary of Mogalakwena River (Limpopo River System). Tributary of Mogol River (Limpopo River System). Tributary of the Pongola River (Pongola River System).
2.7
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
T bk 2: M , rphoDogucall char- cteristics *if g•1cilie barb popullations stuceo d. See T, We 1 for El caHty detaRie.
REFERENCE G1 B.
brevipinnis
G2 B.
brevipinnis
G3 a.
&evil:duals
G4 B.
brevlpinnis
G5 B.
naafi
G6 B.
pallidus
G7 B.
pallidus
G8 a.
pallidus
River Sabie Sand Mogalakwena Mogol Ohrigstad Crocodile Buffets Manzaan
Morphological Characteristics of the Goldie and Chubbyhead Barb Populations Studied
T ?Me 4: Similarry Waccard, 1921 b i - , n pop laticns studied based o meristic characteristics. Sec Tabi 1 for iocaiity det ils.
GI *****
G2 100
G3 100 100
G4 100 100 100 *****
G5 88 88 88 88
G6 88 88 88 88 100 *****
G7 88 88 88 88 100 100
G8 100 100 100 100 88 88 88 ****"
CHI 33 33 33 33 28 28 28 33
CH2 33 33 33 33 28 28 28 33 78 *****
CH5 19 19 19 19 14 14 14 19 68 68
TU1 23 23 23 23 19 19 19 23 45 60 39 *****
TU2 10 10 10 10 7 7 7 10 39 39 60 68
TU3 19 19 19 19 14 14 14 19 52 52 45 88 78
G1 G2 G3 G4 G5 G6 G7 I G8 CHI I CH2 I CH5 TU1 TU2 TU3
2.1
zym z Weres cais Ratliff'
Shortfin Barb (Barbus brevipinnis J b, 1966)
P puiati s with
r nc t descrP d
Barbs Sp ci s from the Transv S • (Loth Afric
CONTENTS
ABSTRACT 3.1
INTRODUCTION 3.1
MATERIALS AND METHODS 3.3
RESULTS 3.4
DISCUSSION 3.5
Figure 1: Map of the Transvaal, depicting the distribution of B.
brevipinnis in relation to the major rivers, as well as the
positions of the sampling points. 3.11
Figure 2: Phylogenetic trees obtained by using: (a) D1SW G and (b)
FREQPARS, showing the relationships between the
populations within the eastern escarpment group (B.
brevipinnis) and the Waterberg group (?B. brevipinnis).
3.12
Table 1: Localities and coordinates where B. brevipinnis (a, b) and
B. ?brevipinnis (C, d) populations were collected. 3.13
Table 2: Enzyme commission numbers (E.G. no) of the proteins
separated by electrophoretic analysis, abbreviations used for
loci resolved and buffers giving best results. 3.14
Table 3: Relative mobilities, allele frequencies and G-test values of the
polymorphic loci and loci where differences were detected
and H values with standard errors for four B.
brevipinnis populations. 3.15
Table 4: Genetic distances between populations calculated with the
distance coefficients: (D = Nei, 1972, D' = Nei, 1978 and C =
Cavelli-Sforza & Edwards, 1967 Chord Distance) for B.
brevipinnis and ?B. brevipinnis. 3.16
Isozyme and Allozyme Differences in Four Shortfin Barb
A ri STRACT
Starch gel-electrophoresis was used to survey genetic variation among samples
from four different populations of Barbus brevipinnis. Thirteen of 30 loci were
found to be polymorphic. It is suggested, based on the extent of the genetic
differences between populations, that the populations collected from the
Waterberg should be regarded as an undescribed species of Barbus.
DATRODUCTBON
Barbus brevipinnis from the Marite River, a tributary of the Sabie River (eastern
Transvaal escarpment), was described by Jubb, (1966). The name refers to the
very short fins found in specimens of this endemic species. Specimens of this
species normally exhibit a dark broken line above the lateral line, consisting of
three to six dashes. A small barb species collected by Gaigher (1969; 1973)
from the upper reaches of the Mogol, Mogalakwena and Lephalala Rivers
(northwestern Transvaal), was initially referred to as B. neefi. However,
subsequent samples collected by Kleynhans (1983) from the same localities
were referred to as B. brevipinnis. Closer inspection of the barbs collected from
the Waterberg revealed that they differed from B. brevipinnis from the Sabie
River in that they displayed a thick solid line above the lateral line, sometimes
consisting of numerous smaller dashes and spots. Examination by Skelton
(pers. comm.) suggested the possibility that the fishes from the Mogol,
Mogalakwena and Lephalala Rivers may belong to a different species.
3.1
Isozyme and Allozyme Differences in Four Shortfin Barb
In terms of the hydrobiological regions as defined by Harrison (1959), B.
brevipinnis from the Sabie River is restricted to the eastern escarpment region
whereas populations from the Mogol, Mogalakwena and Lephalala Rivers occur
in the Transvaal mountain region (Waterberg). The headwaters of these two
regions are completely isolated from one another by a distance of approximately
300 kilometres and are also separated by the Olifants River (Fig. 1).
Although linear measurements were taken from several B. brevipinnis
populations which suggested a larger body size for individuals from the Mogol
River as well as shorter fins in some specimens of B. brevipinnis from the
eastern escarpment region, the determination of the taxonomic status of these
fish populations by means of morphometric and meristic measurements was
found to be inconclusive (Chapter 2).
On the other hand, protein gel-electrophoresis has been instrumental in the
discovery of morphologically cryptic species (Grant et al., 1984). In the present
study the genetic variation within and between B. brevipinnis populations were
investigated to determine whether populations from the eastern escarpment
and the Transvaal mountain regions represent one or more species and to what
extent these populations differ from one another within these two regions.
3.2
Isozyme and Allozyme Differences in Four Shortfin Barb
ATERDALS A ID METH IDS
During September 1990, 60 B. brevipinnis specimens were collected from the
eastern escarpment (Sable and Sand Rivers), and 41 ?B. brevipinnis specimens
from the Mogalakwena and Mogol Rivers (Table 1). The specimens from these
four localities represented the morphological variations observed as discussed
in the introduction. Selected linear and meristic measurements were taken from
all these specimens for comparison, before liver and muscle tissues were
sampled. The muscle tissue of each specimen was individually homogenised in
distilled water. The livers of most specimens were small and groups of five livers
were lumped before analysis. Tissue samples were analysed by starch
gel-electrophoresis according to the methods described by Van der Bank et al.
(1992) and the histochemical methods of Harris and Hopkins (1976) were used
in staining for enzyme activity. The structure of the banding patterns was
deduced from the criteria described by Harris and Hopkinson (1976) and the
locus nomenclature described by Schaklee et al. (1990) was used.
Average heterozygosity (H) was calculated as outlined by Nei (1975). A
G-test for goodness of it was used for non-duplicated loci to determine possible
deviations of allele frequencies from expected Hardy-Weinberg proportions
(Sokal and Rohlf, 1969). The gene diversity analysis (Chakraborty et al., 1982)
was used to test the equality of allelic frequencies among samples within and
between populations. Genetic distances (D=Nei, 1972, D°=Nei, 1978) and C
Cavalli-Sforza and Edwards (1967) chord distance) between selected
3.3
Isozyme and Allozyme Differences in Four Shortfin Barb
populations were calculated using BOOSYS (Swofford and Selander, 1981).
Standard errors of D and D' were calculated according to the methods of Nei
and Roychoudhury (1974). A modification of the Wagner procedure
(FREQPARS), described by Swofford & Berlocher (1987), was used to construct
a phylogenetic tree by applying the principle of parsimony to allele frequency
data, and DISWAG (Swofford and Selander, 1981) was used to construct a
cladogram from Cavalli-Sforza and Edwards (1967) chord distance values.
ES U LTS
Thirty protein coding loci provided interpretable results, of which 23,3%
displayed polymorphism. The enzyme commission numbers, names of the
proteins examined, locus abbreviations and buffers giving the best results are
presented in Table 2. Relative mobilities, allelic frequencies and G-test values
of the polymorphic loci and loci where isozyme and allozyme differences
between populations occurred, as well as H values and standard errors thereof
are presented in Table 3. In summary, all the loci studied were expressed in
muscle tissue although the PGDH and SOD loci were more conspicuous in liver
tissue samples. The AAT, CK, GPI-2, EDIT and DH-2 protein coding loci were
not observed in liver samples. No mobility differences were found in 18 of the
30 loci (AK, ADH, AAT-1 and -2, CK, EST-2 and -3, PROT-1, -2 and -3, GPI-2,
GAPDH, G3PDH, IDDH, LDH-2, DH-1, 7.1 PO, and PEPS) examined and the
alleles at the EST-4 and PROT-4 loci were not detected in the Mogol River
population. Polymorphism was observed only at the EST-1, GPI-1, ODHP,
3.4
Isozyme and Allozyme Differences in Four Shortfin Barb
LDH-1, IMiDH-2, ithIE and PGIVi protein coding loci.
Average heterozygosity values for the Barbus populations studied, based on
thirty loci, ranged from 0.015 to 0.064 (Table 3). The average between
population gene diversity, excluding monomorphic loci, accounted for 77% (±
0.09) of the total diversity leaving only 23% (± 0.09) as the within population
fraction of the total diversity.
Genetic distances (Nei, 1972) between B. brevipinnis and ?B. brevipinnis
populations were greater than 0.240. Smaller genetic distances (0.028 to 0.133)
were found between the populations within these two regions respectively (Table
4). Similar trends were observed for Nei's (1978) and Cavalli-Sforza & Edwards'
(1967) cord distance values between these populations.
The phylogenetic tree (Fig. 2a) obtained by using DISWAG separate the B.
brevipinnis and ?B. brevipinnis populations. However, these groupings were not
obtained by using the FREQPARS procedure (Fig. 2b) because the latter
method produces a completely bifurcating tree, which does not allow two equal
groupings.
DISCUSSION
Differences were observed among populations in both the presence as well as
the frequencies of alternative alleles (Table 3). The two populations from the
3.5
Isozyme and Allozyme Differences in Four Shortfin Barb
Waterberg can be separated from the two escarpment populations based on the
relative mobilities of alleles at the PGDH, SOD and PEPA loci. The EST-4 and
PROT-4 protein coding loci were not detected in the Mogol River population and
the Sand River population could be differentiated from the other populations by
the presence of the PG *110, PG 7,1 *90 and GPI-1*90 alleles. Statistically
significant (P>0.05) deviations of alleles from expected Hardy-Weinberg
proportions occurred at the GPI-1 and a1DH loci for Sand River population
(Table 3). These deviations may have been the result of sampling error since
only 30 individuals could be sampled (Table 1). However, the selection against
a hetro- or homozygote is a common phenomenon within fish populations
(Kirpichnikov, 1981).
Barbus brevipinnis populations are normally isolated in smaller upper
catchment streams as a result of habitat selection which restricts the effective
size of breeding populations. According to Grant and Stahl (1988), species with
small isolated breeding populations will tend to lose alleles and a low H value
is therefore expected in such populations. The lowest values for H would
therefore be expected for the Mogol River population, which is isolated in a part
of a small stream above a waterfall and represents the most restricted of the
populations studied. The Sabie River population, however, reflected the lowest
H value (0.015). The lower H value calculated for this population (Table 3) may
be the result of selection due to siltation and habitat degradation, caused by
forestry and agricultural development in the catchment area of the river.
3.6
Isozyme and Allozyme Differences in Four Shortfin Barb
Mulder (1989) found that the H values of the nine large Barbus species from
southern Africa ranged from 0.052 for B. andrewi to 0.216 for B. mattozi. The
relatively low H value for B. andrewi was attributed to isolation and this value is
comparable with those found in the present study for the Sand and
Mogalakwena River populations. Our estimate is also in agreement with the H
values calculated from the allelic frequencies obtained by Berrebi et al. (1990),
for Saudi Arabian as well as small Barbus species from West Africa (range=
0.028 to 0.278).
The range of D values (0.240 to 0.280) found in the present study between
B. brevipinnis and ?B. brevipinnis populations (Table 4) compare well with D
values reported by Mulder (1989) for nine large Barbus species from South
Africa. Similar D values were also indicated by Agnese et al. (1990) for two small
barbs (0.128) and seven large barbs (0.086 to 0.274). Berrebi et al. (1990)
reported [D values of 0.112 to 0.565 for five small barbs and 0.460 to 1.837 for
three large barb species. Agnese et al. (1990) found D values between two
populations of large Barbus species to be in the region of 0.01. For fish,
Schaklee et al. (1982) found that D values between pairs of conspecific
populations ranged from 0.002 to 0.07 (average 0.05) and for congeneric
species it ranged from 0.03 to 0.61 (average 0.3). Therefore the values of D
(Table 4) between B. brevipinnis and ?B. brevipinnis suggest that these two
groups are congeneric species. This observation is supported by the large (77%)
between-population relative gene diversity compared to the within-population
3.7
Isozyme and Allozyme Differences in Four Shortfin Barb
relative gene diversity fraction (23%) of the total diversity. These differences also
indicate congeneric species rather than conspecific populations. This
assumption is substantiated by pigmentation differences between the
populations as mentioned in the introduction. On the other hand, the values of
D between the B. brevipinnis (0.028) and the ?B. brevipinnis populations (0.133)
are considerably lower, as can be expected for conspecific populations.
The phylogenetic relationship of the barbs in Fig. 2b do not conform to
geological events but the cladogram depicted in Fig. 2a clearly shows the
presence of two groups of barbs, namely a B. brevipinnis and a ?B. brevipinnis
group. The divergent branch lengths (Fig. 2a), in conjunction with the present
distribution pattern of the populations, seems to indicate that ?B. brevipinnis
populated the rivers of the Waterberg region only after the divergence of B.
brevipinnis and ?B. brevipinnis occurred. ?Barbus brevipinnis probably
populated the rivers of the Waterberg region via a linkage between the Limpopo
and the Okavango/Upper-Zambesi Rivers, a migration route suggested by
Gaigher and Poll (1973). According to these authors, many species reached the
Cape via the Nossob and Orange Rivers, including the morphologically similar
B. pallidus and B. neefi which populated the rivers south of the Limpopo River.
The above-mentioned route or stream capturing events on the north eastern
escarpment are the most likely routes whereby the ancestors of B. brevipinnis
could have reached the Sabie River. According to Partridge and Maud (1987),
the capture of the upper Orange River by the lower Orange River dated to the
3.8
Isozyme and Allozyme Differences in Four Shortfin Barb
late Pliocene, which represents a major change in the drainage systems and
could support such a migration route. Based on the assumption that these fish
migrated via the Nossob and Orange Rivers, and that this migration coincided
with the capture of the upper Orange River by the lower Orange River, as
suggested above, the divergence of the Waterberg and eastern escarpment
populations could have happened nearly 3 million years ago (Pliocene).
It was shown that less than 15% of all living organisms are known (according
to some estimates) and that biological systematic research is therefore badly
needed (Raven and Wilson, 1992). Although Bruton (1989) points out that the
adoption of alternative phenotypic states in nature is probably widespread and
that many populations which are currently recognized as species may be no
more than ecophenotypes of one or another homeorhetic state, Scholl (1973)
stated that morphological similarity does not necessarily imply little genetic
variation. The present study has shown that both allozyme and isozyme
differences occur between ?B. brevipinnis and B. brevipinnis (Table 3). They can
thus be viewed as different species, even though little morphological
differentiation has occurred. It is also evident that several genetically unique
populations occur within the two regions which deserve specific conservation
attention. Barbus neefi and B. pallidus, which are morphometrically and
meristically similar to B. brevipinnis (Skelton, pers. comm.), should however be
included in successive studies to determine the phylogenetic relations among
these species. The phylogenetic relationships of populations, representing
3.9
Isozyme and Allozyme Differences in Four Shortfin Barb
species from the major catchment areas, will not only increase our
understanding of the biology and evolution of such species, but may also give
an indication of possible migration routes, as has been demonstrated in this
study.
3.10
Isozvme and Allozyme Differences in Four Shortfin Barb
07 tv
U) to
a)
Co E a)
O
Co
O
O
U)
• cs. . _ (1.)
cci
'Es u)
o) = Q_ ".t..) a)ca3_ =c1
E -- fB co u) co > cn _c C O
I— 2u) CD 0
in 0 0 Q. RS a)
a)
IL
2 -.5
3.11
0 0.4 0.08 0.12 0.16 0.20 0.24 0.28 0.32
SAME (B. brevipinnis)
SAND (B. brevipinnis)
MOGOL (?B. brevipinnis)
MOGALAKWENA (?B. brevipinnis)
Distance from root
DISWAG
(a)
0 1.0
Character state changes FREQ'' ARS
(b)
MOGOL (?B. brevipinnis)
MOGALAKWENA (?B. brevipinnis)
SAND (B. brevipinnis)
SABLE (B. brevipinnis)
Isozyme and Allozyme Differences in Four Shortfin Barb
Figure 2: Phylogenetic trees obtained by using: (a) DISINAG and (b) FREQPARS, showing the relationships between the populations within the eastern escarpment group (B. brevipinnis) and the Waterberg group (?B. brevipinnis).
3.12
Isozyme and Allozyme Differences in Four Shortfin Barb
Table 1: Localities and coordinates where B. brevipinnis (a, b) and ?B. brevipinnis (c, d) populations were collected.
N Locality Latitude Longitude Reference in text
30 Marite Rivera 24°47'S 31 °05'E Sabie River
30 Sand River b 24°09'S 31°02'E Sand River
21 Sterk Rivier c 24°32'S 28°31'E Mogalakwena River
20 Grootspruit d 24°29'S 27°51'E Mogol River
a Type locality (Jubb, 1966), tributary of the Sabie River.
b Major tributary of the Sabie River (Incomati River System).
C Small tributary of the Mogalakwena River (Limpopo River System).
d Isolated population in small tributary of the Mogol River (Limpopo River System).
3.13
Isozyme and Allozyme Differences in Four Shortfin Barb
Table 2: Enzyme commission numbers (E.C. no) of the proteins separated by electrophoretic analysis, abbreviations used for loci resolved and buffers giving best results.
E.C. No Enzyme Locus Buffer
2.6.1.1 Aspartate aminotransferase AAT-1, -2 MF
1.1.1.1 Alcohol dehydrogenase ADH RW
2.7.4.3 Adenylate kinase AK TC
2.7.3.2 Creatine kinase CK RW
3.1.1.1 Esterase EST-1, -2 -3, -4 MF
- - - - General (unidentified) protein PROT-1, -2, -3, -4, -5 MF
Isozyme and Allozyme Differences in Four Shortfin Barb
Table 3: Relative mobilities, allele frequencies and G-test values of polymorphic loci and loci where differences were detected and values with standard errors for four B. brevipinnis populations.
H 0.0146 0.0620 0.0221 0.0462 Standard error ±0.0126 ±0.0295 ±0.0158 ±0.0210
3.15
Isozyme and Allozyme Differences in Four Shortfin Barb
Table 4: Genetic distances between populations calculated with the distance coe icients: (D = Nei, 1972, D° = Nei, 1978 and C = Cavelli-Sforza 81 Edwards, 1967 Chord Distance) for brevipinnis and ? . brevipinnis.
Population
Barbus brevipinnis
Sabie Sand
?Barbus brevipinnis
Mogalakwena Mogol
Sabie D D ' C
Sand D 0.028 D' 0.027 C 0.154
Mogalakwena D 0.240 0.269 D' 0.239 0.268 C 0.412 0.430
Mogol D 0.254 0.280 0.133 D' 0.255 0.279 0.131 C 0.420 0.439 0.334
3.16
Aliozynie differences between pop iations of
hubbyhead barb (Barbus an .plus Weber, 1897)
and Marico barb (B. g otebensis Steindachner,
1894).
CONTENTS:
ABSTRACT 4.1
INTRODUCTION 4.1
MATERIALS AND METHODS 4.2
RESULTS 4.3
DISCUSSION 4.5
Figure 1: Map of the former Transvaal depicting the distribution of B. motebensis and B. anoplus with sampling sites. 4.11
Figure 2:
Phylogenetic trees obtained by using a) DISINAG and b) PAUP, showing the relationship between as well as within populations of B. motebensis and B. anoplus. 4.12
Table 1: Localities where B. anoplus (a, b) and B. motebensis (c, d) populations were collected. 4.13
Table 2: Enzyme commission numbers (E.C. no), proteins examined, abbreviations used for loci resolved and buffers giving best results. Loci nomenclature according to Schaklee et al. (1990). 4.14
Table 3: Relative mobilities (RM), allele frequencies, average heterozygosity (H), exact significance probability values (P) for polymorphic loci and loci where mobility differences were detected between B. motebensis and B. anoplus populations. 4.15
Table 4: Genetic distances between B. motebensis and B. anoplus populations calculated using ID (Nei, 1972), D' (Nei, 1978) and Dc (Cavalli-Sforza and Edwards, 1967). 4.17
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
BSTRACT
Starch gel-electrophoresis was used to assess genetic differences between
two morphologically similar barb species. Two population samples of each
species were analysed and polymorphism was detected in one or both
species, at 10 of the 30 protein coding loci examined. Relative mobility
differences of alleles among the four populations were found at 20 of these
loci (66.7%). It was concluded that the extent of the genetic differences
between the two species supports the present taxonomic status of these
species, which were previously thought to be synonymous. The genetic
differences between the species and populations are of conservation
importance and can be used to study possible migration routes and the
evolution of the species.
1NTR D CTIO
Barbus anoplus is the most widely distributed fish species south of the
Limpopo River and it is mostly limited to altitudes above 915m. Barbus
anoplus was initially described from the Buffels River (Gouritz System) in the
Cape (Jubb, 1968). Morphologically, this species resembles B. motebensis
occurring in some tributaries of the Limpopo River. According to Jubb (1968)
B. motebensis differs from B. anoplus in having a lower caudal peduncle
scale count and the breeding males of the former species exhibit numerous
conical tubercles on the snout, forehead and the lower jaw. Both Gaigher
(1969) and Groenewald (1958) experienced difficulties in separating B.
4.1
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
anoplus from B. motebensis and suggested that the two species are
synonymous. In the present study the genetic variation within and between
four geographically isolated populations were investigated to determine
whether B. anoplus and B. motebensis represent one or more species and
to what extent the various populations differ from each other.
TE
HAILS Ail D ET HODS R
Fifty-six B. anoplus specimens were collected from the Crocodile (Incomati
River system) and Vaal Rivers and 63 B. motebensis specimens were
sampled from the Mario° and Ohrigstad Rivers (Table 1). Figure 1 shows the
distribution of the two species in the former Transvaal and the sampling
points used in this study. Samples were analysed by starch
gel-electrophoresis as described by Engelbrecht and Van der Bank (1994).
Average heterozygosity (H) was calculated according to Nei (1978) and
exact probabilities were used to determine possible deviations of allele
frequencies from expected Hardy-Weinberg proportions (Elston and
Forthofer, 1977; Swofford and Selander, 1981). Different fixation indices
were used to analyse genetic differentiation between populations (Wright,
1978) using 10SYS-1 (Swofford and Selander, 1981): where F IT and F is are
the fixation indices of individuals relative to the total population and its
subpopulations respectively and FsT measures the amount of differentiation
among subpopulations relative to the limiting amount under complete
4.2
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
fixation. Genetic variance was also calculated for each level of hierarchy with
the W - DGHT78 procedure (Swofford and Selander, 1981), using the formula
of Wright (1978). According to Swofford and Selander (1981), this method
is similar to gene diversity analysis used by Nei (1973). The genetic
distances of Nei (1972, 1978), D (standard) and D' (unbiased) respectively
and the Cavalli-Sforza and Edwards' (1967) chord distances (Dc) were
calculated between populations.
Phylogenetic relationships were determined using the DOS G routine
(Swofford and Selander, 1981) with Dc values (phenetic approach) and a
cladogram (cladistic approach) was constructed by phylogenetic analysis
using parsimony (PAUP). The latter procedure uses an allelic frequency data
matrix which is transformed into a presence/absence matrix (an allele
present in sample = 1 and absent = 0). This program is guaranteed to find
the shortest (most parsimonious) tree (Swofford, 1985) and it was preferred
to FREQP RS (Swofford and Berlocher, 1987), because analysis using the
latter method produces a completely bifurcating tree that is confusing when
analysing only four populations to compare the two species.
ES U LTS
The 21 enzymes studied produced interpretable results at 30 protein coding
loci. The enzyme commission numbers, names of the proteins giving
interpretable results, locus abbreviations and buffers giving the best results
4.3
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
are presented in Table 2. Polymorphism was detected at 10 loci (33%) in the
four populations studied and mobility differences of alleles were present at
20 (67%) of the protein coding loci.
The relative allele mobilities at loci where differences between populations
occurred, allelic frequencies and exact significance probabilities for
polymorphic loci, as well as average heterozygosity (H) values and standard
errors are presented in Table 3. All four populations displayed identical allele
mobilities at ADH, AK, G PDH, ODDH, OD1-1', L 2 and PEP Allele
mobility differences separating the two species or the four populations from
one another was detected at the T-1, -2, Cry, EST-I - 4, GPI-I, -2, litfiD11-1-
1, -2, ia1PO, PGDH, PG DA, PROT-I - -5 and SOD protein coding loci.
Relatively low exact significance probabilities for alleles that deviated from
expected Hardy-Weinberg proportions were encountered at 60% of the
polymorphic loci studied (Table 3). Deviations from expected
Hardy-Weinberg proportions were evident at the l E protein coding locus for
all four populations studied; T-1, G 1-2 and Pr-'01--2 for the population
from the Vaal River; EST-I for the Crocodile River population; PEPS for the
Crocodile and Ohrigstad River populations (Table 3). Average heterozygosity
values for the Barbus populations studied ranged between 0.038 and 0.076
(Table 3). F-statistics mean values of -0.008, 0.849 and 0.850 were
calculated for Fps, Fir and FST respectively. The genetic variance values were
4.4
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
6.15, 8.25 and 2.10 for locality-species, locality-total and species-total
analysis respectively.
Genetic distance (D) between the B. anoplus the B. motebensis
populations studied averaged 0.614. Smaller D values (0.230 and 0.329)
were found between the B. anoplus and B. motebensis populations
respectively (Table 4). Values obtained by using various other coefficients
displayed a similar trend (Table 4). The phenetic tree (Fig. 2a) obtained by
using DISVV G, rooted at the midpoint of greatest patristic distance and
based on Dc values (Table 4), illustrates the genetic differences between the
barb populations studied and clearly shows the existence of two separate
groups, namely a chubbyhead (B. anoplus) and a tubercled barb group (B.
motebensis). The cladogram obtained using IPAUIP (Fig. 2b) is almost
identical to the grouping produced by the phenogram (Fig. 2a). This is
probably a result of the high genetic divergence between populations and
the relatively small influence of polymorphic gene frequencies on genetic
distances between these populations.
IDDSCUSSION
Deviations from expected Hardy-Weinberg proportions were encountered at
60% of the polymorphic loci studied (Table 3). Perfect Hardy-Weinberg
populations do not actually exist in nature and departures from
Hardy-Weinberg proportions may occur because of several factors such as
4.5
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
the Wahlund (1928) effect, natural selection, interbreeding and population
bottlenecks (Ferreira et al., 1984). In the present study the deviations from
expected Hardy-Weinberg proportions were mainly caused by a deficiency
of heterozygotes. A deficiency of heterozygotes can be the consequence of
selection against a heterozygote or a homozygote, which is a common
phenomenon within fish populations (Kirpicnikov, 1981). Barbus anoplus
and B. motebensis are mostly confined to the upper catchments of rivers
where natural and artificial barriers often subdivide the species into
numerous isolated populations. It is therefore possible that these deviations
from expected Hardy-Weinberg proportions may be the result of
interbreeding in small and isolated populations, causing a reduction of
heterozygotes (Chakraborty and Nei, 1977).
The H values obtained in the present study (0.038-0.079) are lower than
those found by Mulder (1989) for large Barbus species (0.052-0.216).
However, it compares favourably with the average of H value (0.051) given
by Nevo et al. (1984) for 183 species of fish and by Engelbrecht and Van der
Bank (1994) for small Barbus species. According to Berrebi et al. (1990) and
Agnese et al. (1990) small Barbus species are diploid while the large Barbus
species tend to be tetraploid and it is therefore reasonable to assume that
the relative lower t t values found in certain small Barbus species could be
associated with smaller numbers of active loci in diploids.
4.6
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
The fixation index (F ST), quantifies inbreeding due to population
subdivision or the reduction of heterozygosity of a subdivision due to genetic
drift (Lawson et al., 1989). The FST value (0.850) over all populations
suggests a great genetic differentiation between the populations and is also
comparable with FST values (0.609) found for isolated cave populations of
fish (Avise and Selander, 1972).
The genetic variance is similar to the gene diversity analysis of Nei (1973)
so that the variance of populations in terms of the total (8.25) gives an
indication of total genetic divergence. Most of this divergence is derived from
the variance of the populations compared with the species (6.15), which is
considerably larger than the variance for species compared with the total
variance (2.10). This is indicative of the relative large genetic differentiation
between the four populations and a relative small genetic differentiation
within the populations.
D values ranging between 0.230 and 0.798 where observed in the
present study among the four populations (Table 4) which compares well
with D values reported by Mulder (1989) between nine large Barbus species.
Similar ED values were also found by Agnese et al. (1990) between two
species of small barbs (0.128) and seven species of large barbs (0.086-
0.274). Berrebi et al. (1990) reported values of between 0.112 and 0.565 for
five small barbs. The former author obtained a D value between two
4.7
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
conspecific populations of large Barbus species of approximately 0.01. For
fish, Schaklee et al. (1982) found that D values between pairs of conspecific
populations ranged from 0.002-0.07 (average 0.05) and for congeneric
species it ranged from 0.03-0.61 (average 0.3). According to Grant and Stahl
(1988) the boundaries between taxonomic categories are not sharp but, in
general, the distances between conspecific populations are larger than 0.05
and average about 0.40 for congeneric species. The average value of D
between B. motebensis and B. anoplus (0.614) in the present study falls
within the upper range for congeneric species as discussed above,
supporting the present taxonomic status of separate species. It is likely that
sympatric B. anoplus and B. motebensis communities can occur and that it
could have caused some confusion concerning the specific status of the two
species. The genetic trees (Fig. 2a and b) also depict two groups of
genetically different barbs, namely a chubbyhead barb group (B. anoplus)
and a tubercled barb group (B. motebensis). The comparatively high ID value
(0.329) found between the two B. motebensis populations also falls within
the range for congeneric species, mainly as a result of relative mobility
differences of monomorphic (fixed) alleles at the EST-2, -3 GIPD-1, -2,
MID B1-1, -2 and PROT-5 protein coding loci (Table 3). The presence of these
biochemical markers should be investigated in relation to other
taxonomically associated barb species from southern Africa (e.g. B.
amatolicus and B. gurney!) to determine the taxonomic significance of these
differences. The unexpectedly high level of divergence between the two B.
4.8
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
anoplus populations examined (D=0.230) on the other hand is the result of
mobility differences at fixed alleles at the GPI-1, -2, MPO and PGODH protein
coding loci (Table 3). The taxonomic importance of geographically
subdivided B. anoplus populations have been detected by Barnard (1943),
who divided the chubbyhead barbs into two species, namely B. karkensis
from Natal and B. anoplus for the rest of its distribution. Barnard (1943) also
subdivided B. anoplus into three geographically isolated forms (Orange,
Olifants and Gouritz River Systems) substantiated by morphological
differences among the three forms. The genetic differences between the B.
anoplus populations found in the present study suggest that the
morphological subdivision by Barnard (1943) may be substantiated by a
more detailed study of the genetic differences between these geographically
subdivided B. anoplus populations.
These results also suggest that the dispersion and isolation of these fish
species into the different rivers of southern Africa have created ideal
conditions for speciation. These conditions would result in allopatric
speciation, which is a very common phenomenon in fish populations (Bush,
1975). A subdivided population structure will result in a faster rate of adaptive
morphological evolution and a founder effect in such populations will most
likely lead to genetic changes (Templeton, 1980). According to the latter
author, an adaptive divergence mode of speciation can be present where
populations are divided by intrinsic barriers, as with the present study. Since
4.9
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
the mutation process is random and selection always interacts to some
degree with genetic drift, ordinary micro-evolutionary processes lead to
adaptive divergence between isolated populations even if they inhabit
identical environments. However, the rate of adaptive divergence can be
greatly increased if the environments are also different. The large genetic
variation obtained between the B. anoplus and B. motebensis populations
could therefore be the result of such processes. The ecological isolation of
these species into small isolated populations make these species valuable,
mainly because the genetic variation between the populations can be used
to study micro-evolution, speciation and migration of fish in South Africa.
4.1
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
(1) cs)
U-
Map
of t
he fo
rme
r Tra
nsva
al d
epic
ting
the
dist
ribu
tion
of
B. m
ote
bens
is a
nd B.
ano
plu
s w
ith s
amp
ling
site
s.
4.11
(b)
MARICO (B. motebensis)
CROCODILE (B. anoplus)
VAAL (B. anoplus)
0 1.0
Character state changes PAIR
OHRIGSTAD (B. motebensis)
OHRIGSTAD (B. motebensis) (a)
MARICO (B. motebensis)
CROCODILE (B. anoplus)
VAAL (B. anoplus)
J
0 0.4 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36
Distance from root
DESWAG
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
Figure 2: Phylogenetic trees obtained by using a) DISWAG and b) PAUP, showing the
relationship between as well as within populations of B. motebensis and B. anoplus.
4.12
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
Tab11-1 o LocaDMes where z ant.' .11us (a, L) and la
motel ensis (c, d) p. pull Uons were c , Hect d. .
Species Locality Lat. Long. River
31 B. anoplus Buffelskloofspruit a 24°47'S 30°30'E Crocodile River
25 B. anoplus Blesbokspruit b 26°11'S 28°23'E Vaal River
33 B. motebensis Ohrigstad River c 24°53'S 30°36'E Blyde River
30 B. motebensis Kaaloog se Loop d 25°47'S 26°24'E Marico River
a Tributary of the Crocodile River (Incomati River System).
b Tributary of the Vaal River (Orange River System).
Tributary of the Olifants River (Limpopo River System).
d Tributary of the Marico River (Limpopo River System).
4.13
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
Table 2: Enzyme Commission Numbers, proteins examined, abbreviations used for loci resolved and buffers giving best results. Loci nomenclature according to Schaklee et al. (1990).
E.C. No Enzyme Locus Buffer
2.6.1.1 Aspartate aminotransferase AAT-1, -2 MF
1.1.1.1 Alcohol dehydrogenase ADH RW
2.7.4.3 Adenylate kinase AK TC
2.7.3.2 Creatine kinase CK RW
3.1.1.1 Esterase EST-1, -2 , -3 MF
- - - - General (unidentified) protein PROT-1, -2, -3, -4, -5 MF
Table 3: Reiative rnobiiities (RI I), allele frequencies, average heter zyi osity ( , ex ct significance probabilities values (P) for polymorphic loci and loci where mobility differences were detected betw en 11_3 motebensis and °plus populations.
H 0.044 0.038 0.044 0.079 a ± 0.024 ± 0.023 ± 0.023 ± 0.029
4.16
Allozyme Differences Between Populations of Barbus anoplus and B. motebensis
Table 4: Genetic distances between 0. motebensis and z. anoplus populations calculated using D ( ei, 1972), Du( Nei, 1978) and roc (Cavalli-Sforza nd Edwards, 1967).
Barbus motebensis Barbus anoplus
Ohrigstad Mario° Crocodile Vaal
Mario° River
D 0.329 -
D' 0.328 -
Dc 0.470 -
Crocodile River
D 0.798 0.660 -
D' 0.797 0.659 -
Dc 0.658 0.616 -
Vaal River
D 0.575 0.424 0.230 -
D' 0.574 0.423 0.228 -
Dc 0.596 0.530 0.416 -
4.17
Genetic relationships betw. n Mari= barb
(Barbus motebensis Steindach r, 1! 4) 9 'edtaH
barb (B. gum yi G nth r9 186 ) a d A atoL barb
(B. amatoiicus Skeito 199 ).
C NTENTS:
ABSTRACT 5.1
INTRODUCTION 5.1
MATERIALS AND METHODS 5.4
RESULTS 5.6
DISCUSSION 5.7
Figure 1: Map of southern Africa depicting the distribution of B.
motebensis, ?B. motebensis, B. gurneyi and B. amatolicus
with sampling sites. 5.14
Figure 2: Phenetic tree obtained by using DHSPAN and D showing
phylogenetic relationship between B. motebensis, ?B.
motebensis, B. gumeyi and B. amatolicus. Bootstrap numbers
system (Ridgway et al., 1970). TC: continuous Tris, citric acid (pH 6.9) buffer system (Whitt, 1970).
5.16
Table 2: r=elative mobilities, all& frequencies, verage heterozygosity (H), exact significa ce probability values (F) for polymorphic loci and loci where mobility differences were detected between four t arbus populations.
Locus Allele ? B. motebensis B. motebensis B. amatolicus B. gurney!
H 0.044 0.037 0.110 0.015 a ± 0.024 ± 0.023 ± 0.040 ± 0.015
5.18
Genetic Relationships Between Barbus motebensis, B. gumeyi and B. amatolicus.
Table 3. Genetic distances between ? . motebensis, motebensis, gurneyi and . amatolicus populations calculated using D (Nei, 1972), D' (Nei, 1978) and Dc (Cavelli-Sforza & Edwards, 1967).
. motebensis 1. motebensis matolicus
. motebensis
D
D'
Dc
0.343
0 .342
0.478
B. amatolicus
D 0.375 0.398
D' 0.368 0.390
Dc
gumeyi
0.501 0 .506
D 0.296 0.395 0.252
D' 0.295 0.393 0.245
Dc 0.453 0.509 0.422
5.19
Genetic relationships between sever sp cies
withi the chap • byh d d godc os a groups of
osces 9 ro i m ida ).
CO TE TS
ABSTRACT 6.1
INTRODUCTION 6.2
MATERIALS AND METHODS 6.4
RESULTS 6.5
DISCUSSION 6.7
Figure 1: Phylogenetic tree, using =PAN and Nei's (1978) values,
depicting relationships within and between the goldie,
chubbyhead and tubercle barb groups of minnows studied.
Bootstrap numbers are listed at nodes and population
designations are presented in Table 1. 6.18
Table 1: Sample size, species and localities where populations of
chubbyhead and goldie barbs were collected. Species listed
according to existing taxonomy. 6.19
Table 2: Enzyme commission numbers (E. C. No.), proteins examined,
abbreviations used for loci resolved and buffers giving best
results. 6.20
Table 3: Relative mobilities (Rilli), allele frequencies, average
heterozygosity (H), exact significance probability values ( ) for
polymorphic loci and loci where mobility differences were
detected between chubbyhead, tubercle and goldie barb
population. See Table 1 for abbreviations of populations. 6.21
Table 4: Genetic distance values (Nei, 1972) between populations of
chubbyhead, tubercle and goldie barbs. See Table 1 for
abbreviations of populations. 6.23
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
ABSTRACT
The chubbyhead and goldie barbs are the most widely distributed groups of
minnows in the cooler rivers of southern Africa, south of the Limpopo River. The
morphological similarity between species within these two groups led to
uncertainty regarding the taxonomy, distribution and conservation status of the
different species. Consequently, conservation bodies experienced difficulties to
describe the variation within these two groups through the existing taxonomy and
to determine the conservation status of the different species. To contribute
towards solving this problem, starch gel-electrophoresis was used to assess the
genetic differences between populations and species within these two groups of
minnows.
The result of the present study suggests that a third group of barbs exist,
splitting the present chubbyhead group of barbs into a tubercle and a
chubbyhead group. Furthermore, the chubbyhead barbs can be subdivided into
at least two groups consisting of a eastern CapeNaal River group and a north-
eastern escarpment group (Crocodile and Tugela Rivers). The present study
also suggests that the goldie barbs consist of a B. brevipinnis group from the
north-eastern escarpment (Sabie and Pongola Rivers), a goldie barb group from
the rivers of the Waterberg (north-western area of high topographical elevation
in the catchment of the Limpopo River), and a goldie barb group from the
eastern interior (Ohrigstad, Crocodile and Tugela Rivers). The genetic distances
between three species of tubercle barbs (B. motebensis, B. gumeyi and B.
6.1
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
amatolicus) and a population of tubercle barbs from the Ohrigstad River (mean:
0.329) suggest that the latter population may represent a fourth species of
tubercle barb endemic to the Steelpoort and Blyde Rivers. Besides the
taxonomic and biogeographic implications of this study, it also provides
evidence that B. neefi was probably recently translocated between catchments.
BBB DUCTBON
Describing existing diversity of organisms in terms of their spatial and temporal
distribution and abundance is essential for the conservation of biotic diversity.
In southern Africa, researchers have been mainly involved in studies
surrounding the classification and distribution of freshwater fish species based
on their morphological characteristics. (Gilchrist and Thompson, 1913; Barnard
1973, 1976; Kleynhans, 1983; Skelton, 1993). The need for phylogenetic
studies of freshwater fish species is highlighted by an extensive history of
uncertainty regarding the taxonomy of the different species of goldie and
chubbyhead barbs in these studies. Because of these uncertainties, it is not
always possible for conservation bodies to determine whether a species within
these two groups is common, rare or threatened. For example, Skelton et al.
(1995) lists the conservation status of B. motebensis as "status not known
sufficiently".
The chubbyhead barbs are endemic to South Africa (Skelton, 1993) and
6.2
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
according to present taxonomy, they comprise the chubbyhead barb (Barbus
anoplus), the redtail barb (B. gurney!), the Marico barb (B. motebensis) and the
Amatola barb (B. amatolicus). Tubercles in the males of the latter three species
set them apart from B. anoplus (Skelton, 1993). Chubbyhead barbs are widely
distributed in the headwaters of many rivers south of the Limpopo River.
Pigmentation in the chubbyhead barbs is usually simple, consisting of an
indefinite body stripe and a small spot on the base of the caudal fin. This group
of minnows are of ecological and scientific interest because they are frequently
the only fish species in many of these rivers and are often found in small
isolated stretches of river above waterfalls.
The "goldie" barbs are also a distinct southern African group of minnows and
comprise the goldie barb (B. pallidus), the shortfin barb (B. brevipinnis) and the
sidespot barb (B. neefi). Goldie barbs are relatively small barbs and
pigmentation usually consist of spots, dashes or a single stripe along the
midbody. The goldie barbs are limited to the headwaters of rivers and
occasionally occur with chubbyhead barbs.
The morphological similarity of the species, the difficulties encountered
separating them from each other and attempts to decide their phylogenetic
relationships showed some inconsistencies in terms of their present taxonomic
status. Therefore, a reliable method independent of morphological
characteristics was required to define the species within these groups. The
6.3
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
computation of genetic distances based on data from several loci has been
successfully used to evaluate the taxonomic relationship between Cyprinidae
(Berrebi et al., 1990; Agnese et al., 1990; Cook et al., 1992; Alves and Coelho,
1994; Engelbrecht and Van der Bank, 1994; Coelho et al., 1995; Karakousis et
al., 1995; Machordom et al., 1995). In the present study the phylogenetic
relationships between seven species (17 populations) were investigated to
determine their taxonomic relationships.
MATERIALS AND METHOL S
Three hundred and thirty Barbus specimens were collected from 17 localities
within South Africa (Table 1). Samples were analysed by starch
gel-electrophoresis as described by Engelbrecht and Van der Bank (1994).
Average heterozygosity (H) was calculated according to Nei (1978) and exact
probabilities were used to determine possible deviations of allele frequencies
from expected Hardy-Weinberg proportions (Elston and Forthofer, 1977;
Swofford and Selander, 1981). Genetic variances were calculated for each level
of hierarchy with the WRDGHT78 procedure (Swofford and Selander, 1981).
According to Swofford and Selander (1981), this method is similar to gene
diversity analysis (Nei, 1973). The genetic distances of Nei (1972, 1978), D and
D' respectively were calculated between populations. The D value was used
for comparisons with other studies. However, D' is a more appropriate measure
for small sample sizes and was used for determining phylogenetic relationships.
6.4
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
D1SP N (Copyright: Tatsua Ota, 1993, Pennsylvania State University, USA)
was used to construct a phylogenetic tree from D ° values, using neighbour-
joining and bootstrap tests (1000 replications). Phylogenetic relationships were
also determined, using a phenetic approach from D ° values, with the CASTE
and =WAG routines (Swofford and Selander, 1981), and a cladistic approach
using F EQPA (.•1) S (Swofford and Berlocher, 1987) which produces a
completely bifurcating tree.
ESULTS
Twenty-one proteins were studied which produced interpretable results at 30
protein coding loci, of which 53% displayed polymorphism. The enzyme
commission numbers of the proteins, locus abbreviations and buffers giving the
best results are presented in Table 2. Five of the loci ( DH, AK, GAPDH, DDDH
and ILDH-2) displayed monomorphic gel banding patterns. Mobility differences
of alleles between populations studied were present at 22 (73%) of the protein
coding loci studied. Products of the
T-2, EST-3 and P
T-5 protein coding A •
loci migrated cathodally. Products of the SOD protein coding locus in the four
tubercle barb populations (TU1-4 in Table 1) also migrated cathodally whereas
it migrated anodally in all of the other populations studied.
The relative allele mobilities at loci where differences between populations
occurred, allelic frequencies and exact significance probabilities for polymorphic
loci, as well as average heterozygosity (H) values and standard errors thereof
6.5
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
are presented in Table 3. For polymorphic loci, low exact significance
probabilities of alleles which deviated from expected Hardy-Weinberg
proportions were encountered in 48%, 58% and 100% of the cases within the
chubbyhead, tubercle and goldie barb populations studied respectively (Table
3). A deficiency of heterozygotes was indicated in 87% of the cases where
deviations from expected Hardy-Weinberg proportions occurred. As indicated
in Table 3, an excess of heterozygotes was only present at the ME protein
coding locus for three of the chubbyhead (CH1, CH2, and CH5) and tubercle
barb (TU1, TU3 and TU4) populations respectively, and at the ODHP protein
coding locus in two of the goldie barb populations (G5 and G6). Average
heterozygosity values for all the populations studied, based on 30 protein
coding loci, ranged between 0.015 and 0.110 (Table 3).
Values of ID° differed from ID values only at the second decimal. Since these
values were almost similar, reference is made to D values to compare the
results of the present study with those of other authors. The mean ID values
between the chubbyhead - tubercle, chubbyhead - goldie and tubercle - goldie
barb groups were 0.706 (range: 0.424-1.052), 0.633 (range: 0.449-0.907) and
1.006 (range: 0.770-1.247) respectively. The mean ID values between species
and/or populations within these three groups were 0.221 (range: 0.111-0.404),
0.328 (range: 0.242-0.380) and 0.182 (range: 0-0.293) for the chubbyhead,
tubercle and goldie barb groups respectively (Table 4).
6.6
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
The phylogenetic tree (Fig. 1) obtained by using DOSPAN and DU values
(Table 4) illustrates the genetic relationships between the barb populations
studied and depicted the existence of three main groups consisting of a
chubbyhead, a tubercle and a goldie barb group. The present study found
genetic distances of the same magnitude between three species of tubercle
barbs (B. motebensis, B. gurneyi and B. amatolicus) and a population of
tubercle barbs from the Ohrigstad River (?B. motebensis) and is depicted as a
separate species in Fig. 1. The chubbyhead barbs are subdivided into at least
two groups consisting of a group from the Vaal River (CH5) and rivers of the
eastern Cape (CH3 and CH4), and a group from the Crocodile (CH2) and
Tugela Rivers (CH1). The clustering also suggests that the goldie barbs consist
of a B. brevipinnis group from the north-eastern escarpment (G3 and G4), a
goldie barb group from the Waterberg (Mogol and Mogalakwena Rivers), and
a goldie barb group from the Ohrigstad (G5), Crocodile (G6) and Tugela Rivers
(G7). This grouping is almost identical to those obtained by using FREQP A RS,
CLUSTER and MS G with DU values. A hierarchy based on these groupings
(three main groups with nine specie-groups) gave genetic variance (Wright,
1978) values of 2.86, 11.29 and 8.43 for population to species-groups,
population to total and species-groups to total analysis respectively.
DISCUSSI
Allelic frequencies (Table 3) deviated from expected Hardy-Weinberg
proportions at a relative large number of the polymorphic loci studied (48%,
6.7
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
58% and 100% for chubbyhead, tubercle and goldie barb groups respectively).
Departures from Hardy-Weinberg proportions may occur because of several
factors such as the Wahlund (1928) effect, natural selection, interbreeding and
population bottlenecks (Ferreira et al.,1984). In the present study the deviations
of allele frequencies from expected Hardy-Weinberg proportions were mainly
caused by a deficiency of heterozygotes. A deficiency of heterozygotes can be
the consequence of selection against a heterozygote or a homozygote, which
is a common phenomenon within fish populations (Kirpicnikov, 1981).
Chubbyhead, tubercle and goldie barbs are mostly confined to the upper
catchments of rivers where natural and artificial barriers often subdivide the
species into numerous isolated populations. It is therefore possible that the
observed deviations of allelic frequencies from expected Hardy-Weinberg
proportions may be the result of interbreeding in small and isolated populations,
causing a reduction of heterozygotes (Chakraborty and Nei, 1977).
The H values obtained in the present study (range: 0.015-0.110) are slightly
lower than those reported by Mulder (1989) for large Barbus species (range:
0.052-0.216). However, it is similar to the H values given by Nevo et al. (1984)
for 183 species of fish, Alves and Coelho (1994) for a cyprinid species and by
Engelbrecht and Van der Bank (1994, 1996, submitted') for small Barbus
species. According to Berrebi et al. (1990) and Agnese et al. (1990) small
Barbus species are diploid while the large Barbus species tend to be tetraploid
and it is therefore reasonable to assume that the relative lower H values found
•
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
in small Barbus species could be associated with smaller numbers of active loci
in diploids. The highest heterozygosity values in the present study were
observed in populations sampled from the eastern Cape (TU4, CH4 and CH5),
which may suggest a possible relation between genetic diversity and this
specific habitat. This may also be related to extensive breeding (Pirie Trout
hatchery) and stocking of minnows in these rivers as trout fodder (Skelton, pers.
comm). In contrast to these, the low heterozygosity values obtained (i.e. TU3
and G1) may be the result of environmental degradation. This assumption is
supported by the fact that some of these populations were heavily infested with
parasites, showing possible stress. Alves and Coelho (1994) emphasized that
habitat degradation will inevitably lead to the reduction of intraspecies genetic
diversity.
The phylogenetic tree (Fig. 1) obtained in the present study give plausible
groupings that could contribute towards understanding the taxonomy of the
chubbyhead and goldie barb groups (three main groups and nine specie-
groups). Because the taxonomy of the species has not yet been properly
settled, this phylogenetic clustering of the different populations was used to
deduce species-groups. Based on these three main groups and nine species-
groups, it was found that most of the total genetic variance (11.29) is derived
from the species-groups towards the total (8.43), which is much larger that the
variance for populations in terms of the species-groups (2.86). This shows the
relative large genetic differentiation between species-groups and much less
differentiation between populations within these species-groups. This
6.9
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
delineation (Fig. 1) is therefore viewed as representative of the genetic
relationship between the populations studied. This delineation is also supported
by distribution patterns and morphological characteristics of the populations
within these species-groups (see discussion below).
Phyllogenetic subdivision of chubbyheads into a chubbyhead group and
tubenclle barb group
The most important division in Fig. 1 is the split between the chubbyhead group
and the tubercle barb group which were previously thought to be closely related
(Skelton, 1993) or synonymous (Groenewald, 1958; Gaigher, 1969, 1973,
1976). The allele mobility differences at the .A T-1 (see Fig. 2 in Appendix), -2,
Clcc, EST-1, GDH, P OT-1 - -3 and SOD protein coding loci separated the
tubercle barb group from the chubbyhead barb group (Table 3). The D value
between these two groups averaged 0.706 (range: 0.424-1.052). For fish,
Shaklee et al. (1982) found that D values between pairs of conspecific
populations ranged from 0.002-0.07 (average 0.05) and for congeneric species
it ranged from 0.03-0.61 (average 0.3). According to Grant and Stahl (1988) the
boundaries between taxonomic categories are not sharp. Usually the distances
between conspecific populations are larger than 0.05 and average about 0.40
for congeneric species. The average value of D in the present study between
chubbyhead and tubercle barbs (0.706) falls within the upper range for
congeneric species as discussed above, supporting the present taxonomic
status of separate species. Because of evolutionary age differences between
6.10
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
taxa, generalised average D values between congeneric species are not
necessarily reliable. However, comparisons with closely related taxa could be
useful.
According to Thorpe and Sole-Cava (1994) and Karakousis et al. (1995)
genetic divergence can be related to evolutionary time. Therefore the large
genetic differences found in the present study between the different species
and populations of chubbyhead and tubercle barbs suggest that these
populations have been isolated from each other for millions of years. In view of
these large genetic differences and the overlapping distribution patterns of
these two groups, the possibility that these two groups have their origin in
different lineages and that their morphological similarity is the result of
convergence should not be excluded.
Several systematic studies of the Cyprinidae which have used tuberculation
as a primary character to differentiate species-groups, species and subspecies
(Gibbs, 1957; Lachner, 1967; Skelton, 1988). The presence of tubercles in the
tubercle barbs would substantiate a split between the chubbyhead and tubercle
barbs. Therefore, the presence of tubercles in the tubercle barb group (B.
gurney!, B. motebensis and B. amatolicus) clearly differentiates these species
from the chubbyhead barb (B. anoplus). The former two species (B. gurney! and
B. motebensis) also differ from B. anoplus in having a lower caudal peduncle
scale count and two pairs of barbels (Jubb, 1968; Skelton, 1993). Barbus
6.11
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
amatolicus, which occur sympatrically with B. anoplus, remained undetected
until recently because of the subtle differences between the two species, but its
inferior mouth (compared with a terminal mouth), tubercles in males and one
pair of well-developed barbels set it apart from B. anoplus (Skelton, 1990). The
phylogenetic differences between B. anoplus and B. motebensis are discussed
in more detail by Engelbrecht and Van der Bank (submitteda).
Phyiogenetic subdivision of the tubercle barb group of minnows
Engelbrecht and Van der Bank (1996) discussed the phylogenetic relationships
between three species of tubercle barbs (B. motebensis, B. gurneyi and B.
amatolicus) and a population of tubercle barbs from the Ohrigstad River, and
showed that the latter population may represent a fourth species of tubercle
barb endemic to the Steelpoort and Blyde Rivers. The comparatively high D
value (0.327) calculated between the B. motebensis population (TU2) and the
tubercle barb population from the Ohrigstad River (TU1), which were previously
classified as B. motebensis, is similar to
values obtained between three I)
species of tubercle barbs and falls within the range predicted for congeneric
species. This division is mainly caused by fixed allele differences at the
following monomorphic loci:
T-2, EST-2, -3, GPi-1, -2 (see Fig. 3 in AA
Appendix), DH-1, -2 and PROT-5 (Table 3).
The present study has shown that the tubercle barb population from the
Ohrigstad River (TU1) is phylogenetically closest to the B. gurneyi (TU3)
6.12
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
population (Figure 2). Morphological similarities between the tubercle barb from
the Ohrigstad River (TU1) and B. gumeyi (TU3), which divide them from B.
motebensis (TU2), is the presence of only two to three scales between the
complete lateral line and the ventral fin in comparison with four scales and an
incomplete lateral line in the latter species. However, the D value (0.282)
between the tubercle barb population from the Ohrigstad River (TU1) and B.
gurneyi (TU3) also falls within the range for congeneric species and is mainly
the result of mobility differences at the EST-I, -3,
Dll-I-1,liiiiP0 and IPG^Y1 protein m
coding loci. The first reference to this tubercle barb species in the north-eastern
escarpment was made by Crass (1960, 1964), who considered barb specimens
collected from a small tributary of the Sabie River (which is in the proximity of
the Ohrigstad River) comparable to B. gurneyi.
Phylogenetic subdivision of the chubbyhead barb group of minnows
Based on the result of the present study (Fig. 1) the existing chubbyhead group
of barbs are subdivided into at least two groups: B. anoplus from the Vaal River
and eastern Cape rivers, and ?B. anoplus from the Crocodile and Tugela Rivers
(north-eastern escarpment). The mean value of
(0.274) between these two [I)
groups falls within the range for congeneric species and is mainly supported by
relative mobility differences of alleles at the GR-2, PGDFI (see Fig. 4 in
Appendix), PROT-1 and -2 protein coding loci. This subdivision agrees largely
with Barnard's (1943) suggestion to subdivide the chubbyhead barbs into two
species namely B. karkensis from Natal, and B. anoplus from the rest of its
6.13
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
distribution. The most obvious morphological difference found in the present
study between these two groups is the presence of an incomplete lateral line
in the Vaal River/eastern Cape group and a complete lateral line in the north-
eastern escarpment group. The latter group also lack the distinctive
"chubbyhead" profile. These differences are discussed in more detail by
Barnard (1943) and Gaigher (1969, 1973, 1976).
Phyiogenetic subdivision of the goldie barb group of rninnows
The goldie barbs are depicted in Fig. 1 as a group separate from the
chubbyhead and tubercled barbs. This is supported by relative mobility
differences of alleles at the LIDF0-1 (see Fig. 1 in Appendix), « E, PGDH, PROT-
1 and SOID protein coding loci. Although Skelton (1993) divided the goldie barbs
into three species, he mentioned that the taxonomy of the goldie barbs has not
yet been properly settled. According to this division the goldie barbs consist of
B. pallidus which has a divided distribution in the coastal streams of eastern
Cape, and highveld tributaries of the Vaal, Crocodile, Steelpoort, Pongola and
Tugela Rivers; B. neefi with a divided distribution in the Steelpoort River
(Limpopo River System), and Upper Zambezi, Kafue Rivers and rivers of
southern Zaire; B. brevipinnis with a divided distribution in the Sabie River
(Incomati River System), and rivers from the Waterberg. Although the latter
distribution was not referred to in the publication mentioned above, material
from this locality was referred to as B. brevipinnis by the same author.
6.14
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
Based on the result of the present study (Fig. 1), the goldie barbs were
divided into three groups that differ slightly from these groupings. Group 1
consists of the Sabie, Sand and Pongola River populations (G1, G2 and G8).
It is believed that this group represents the species B. brevipinnis because G1
was sampled close to its type locality. It is interesting that the Pongola River
population (G8) was classified as B. pallidus during sampling (Table 1).
Group 2 consists of the Ohrigstad, Crocodile and Tugela River populations
(G5, G6 and G7), which suggests that the B. pallidus and B. neefi populations
studied are conspecific populations and not congeneric species. However, B.
pallidus and B. neefi were not sampled from the vicinity of their type localities
for the present study (eastern Cape coastal streams and headwaters of the
Zambezi River respectively). These localities are geographically completely
separated from the populations studied and may represent two distinct species-
groups. This conclusion must be confirmed by further studies. The D value
between populations G5 and G6 in the present study was zero, which is unlikely
for two congeneric species completely isolated from each other in different river
systems (Crocodile River in the Incomati System and Ohrigstad River in the
Limpopo River System). In comparison, the D value between these two
populations and G7 were 0.125 and 0.128 respectively. Agnese et al. (1990),
Machordom et al. (1995) and Cook et al. (1992) obtained D values ranging from
0.001-0.01 between conspecific populations of cyprinids (including Barbus).
Therefore, it is believed that the low D value obtained between these two goldie
6.15
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
barbs (G5 and G6) show conspecific population variation and that these fish
could have been accidentally translocated from the Lydenburg Fisheries Station
to these localities, which would explain the genetic similarity between the two
populations. This hypothesis is supported by the fact that Gaigher (1969, 1973)
did not sample goldie barbs during their surveys in the Crocodile River (G6),
and that B. neefi (G5) was collected for the first time in the Ohrigstad River
during the mid-sixties (Jubb, 1967, 1968).
Group 3 consists of the Mogol and Mogalakwena River populations (G3 and
G4). Engelbrecht and Van der Bank (1994) suggested that this group may
represent a new species and that it may be limited to the rivers of the
Waterberg. Although the subdivision of group 1 and 2 are supported by a
relative low bootstrap value (38), this subdivision is also supported by
morphological differences between specimens from populations G5 (group 2)
and G1 (group 1). These differences include a shorter head with neurocranium
more curved, narrower ethmoid and infraorbital bone in the former species
(Skelton, pers. comm.).
The present study contributed towards understanding the phylogenetic
relationships, taxonomy and distribution (artificial and natural) within the
chubbyhead and goldie barb groups of minnows, which has troubled
researchers for decades (Barnard, 1938, 1943; Groenewald, 1958; Crass,
1960, 1964; Gaigher, 1969, 1973, 1976; Skelton, 1993). This study is yet
another illustration that molecular methods can be very useful for the study of
6.16
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
(conspecific) populations and morphologically very similar species. The relative
large D values between most of the populations studied suggests that most of
these populations have been isolated from each other for considerable time in
habitats which differ and that genetic divergence within and between species
have taken place. The results of this study also suggest that the distribution of
some species is more limited than previously thought (i.e. B. motebensis and
B. brevipinnis) and their status may be rare. However, the most important
conclusion drawn from these results is that the species-concept is a poor
measure of ecological important diversity because species are phylogenetically
not always clear cut, but could contain a continuum of genetically-unique
populations. It is known that conservation priorities are often driven by the
presence of rare and endangered species. These priorities often ignore
genetically-unique populations, which could be rare or even endangered.
Therefore, conservation priorities should perhaps not be bound by species and
species diversity, but should also strive to conserve genetic diversity within
common and often ecologically important populations such as within the
chubbyhead, tubercle and goldie barbs.
6.17
YCJ =
Iii ■1
),- U 14 , at .0., gt VI E co CO co 1.1.1 03 .-I ad = Ce 03 CC 0 < = < = < 0 CO U CO i••• CO
V
V co
te•■=.==.3
r-
U
U 1.1
U
U
U
co V
7
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
a) u) .c a) O
CD C a) 4—
CZ
..;:a)
02 c - (L)
(1) E a_ YE c 2 c-
E2 ca -(7; 2 8 moo
0 a) a La a) z
"C:3
U)- .
O r' Ct) C CD
C
N. 4— o IA
eL
z a) C
O 9.2 < E.2 co CL a) co
c . o
cm a C C c c -(7) co ,D) = 7) (L)- -CO
_c c 0 _a .2
j3 76 c a) cf.. :0 o
0— CI) ca
6.18
Genetic relationships between seven species within the chubbyhead and goldie barb groups of minnows
Table 1: Sat ple size, species and localities where populations of chubbyhead annd goldie rbs were collected. Species listed ccordi.r g to existing taxonomy.
N Species Locality Latitude Longitude Abbreviation
31 10 25 10 10
33 30 10 10
30 30 21 20
10 10 30 30
CHUBIBYHEAD BAR IS
B. anoplus Buffelskloofspruit A 24°47'S 30°30'E CH2 B. anoplus Ngagagane B 27°59'S 29°52'E CH1 B. anoplus Blesbokspruit C 26°11'S 28°23'E CH5 B. anoplus Gatbergvlei ° 31°15'S 28°04'E CH3 B. anoplus Bloukrans River E 33°19'S 26°33'E CH4
B. motebensis Ohrigstad River F 24°53'S 30°36'E TU1 B. motebensis Kaaloog se Loop ° 25 ° 47'S 26°24'E TU2 B. gurneyi Msinduzi H 29°38'S 30°25'E TU3 B. amatolicus Xuka River' - 31 °28'S 27°51'E TU4
GOLDIE BARBS
B. brevipinnis Marite River' 24°47'S 31°05'E 01 B. brevipinnis Sand River " 24°09'S 31 °02'E G2 B. brevipinnis Sterk River '' 24°32'S 28°31'E G4 B. brevipinnis Grootspruit I' 24°29'S 27°51'E G3
B. pallidus Manzaan River N 27°38'S 30°53'E G8 B. pallidus Ngagagane B 27°59'S 29°52'E G7 B. pallidus Buffelskloofspruit A 24°47'S 30°30'E G6 B. neefi Ohrigstad River F 24°53'S 30°36'E G5
A B c D E F G H 1 , K L M N
Tributary of the Crocodile River (Inkomati River System). Tributary of the Buffels River (Tugela River System). Tributary of the Vaal River (Orange River System). Tributary of Inxu River (Mzimvubu River System). Tributary of the Cowie River (Cowie River System). Tributary of the Olifants River (Limpopo River System). Tributary of the Marico River (Limpopo River System). Tributary of the Mgeni River (Mgeni River System). Tributary of the Xuka River (Mbashe River System). Type Locality (Jubb, 1966), Tributary of Sabie River (Incomati River System). Tributary of Sabie River (Incomati River System). Tributary of Mogalakwena River (Limpopo River System). Tributary of Mogol River (Limpopo River System). Tributary of the Pongola River (Pongola River System).
6.19
Genetic relationships between seven species within the chubbvhead and goldie barb groups of minnows
Table 2: Enzyme commission numbers (E. C. no), proteins exami ed, abbreviations used for loci resolved and buffers giving best results.
E.C. No Enzyme Locus Buffer
2.6.1.1 Aspartate aminotransferase AAT-1, -2
1.1.1.1 Alcohol dehydrogenase ADH
2.7.4.3 Adenylate kinase AK
2.7.3.2 Creatine kinase CK
3.1.1.1 Esterase EST-1, -2 , -3
- - - - General (unidentified) protein PROT-1, -2, -3, -4, -5
Genetic relationships between seven species within the chubbvhead and qoldie barb groups of minnows
Table 3: Relative mobilities (RM), allele frequencies, average heterozygosity (H), exact significance probability values (P) for polymorphic loci and loci where mobility differences were detected between chubbyhead, tubercle and goldie barb population. See Table I for abbreviations of populations.