Colour forms of Amazonian cichlid fish represent reproductivelyisolated species

J. S. READY,*� I . SAMPAIO,� H. SCHNEIDER,� C. VINSON,� T. DOS SANTOS�& G. F. TURNER�*Department of Vertebrate Zoology, Swedish Museum of Natural History, Stockholm, Sweden

�Department of Biological Sciences, University of Hull, Hull, UK

�Federal University of the State of Para, Braganca, PA, Brazil

Introduction

Systematists working on cichlid fishes from the species-

rich East African Lakes have often described colour

forms as different species (McKaye et al., 1984; Arnegard

et al., 1999), and this has a major influence on the

estimated species richness of groups of these fishes

(Genner et al., 2004). Amongst closely related haplo-

chromine cichlids in Africa both allopatric and sympatric

species pairs frequently demonstrate prezygotic isolation

(Seehausen et al., 1997; Knight et al., 1998; Turner et al.,

2001), even when post-zygotic isolation is absent

(Seehausen et al., 1997). Amongst closely related hapl-

ochromine cichlids in Africa, only a few cases of post-

zygotic isolation have been reported, and are generally

between anciently diverged allopatric species (Turner

et al., 2001).

For South American cichlids, melanic patterns are

generally used for taxonomic description but other

pigments, which do not preserve as well are not usually

used (Kullander, 1980; Kullander & Silfvergrip, 1991).

The reproductive behaviour of many species has been

described in hobbyist literature (e.g. Richter, 1988;

Koslowski, 2002; Staeck, 2003), where it is reported that

colour forms hybridize freely. As a result allopatric colour

forms have been generally assumed to be conspecific

when no other characters have been found to distinguish

them. However, there is no indication from such anec-

dotal observations that a choice of a mate from the same

population was available. The use of no choice trials may

reflect the options of a single immigrant female into

another population, but the results would not argue

against species status. Experiments with African cichlids

have shown that many good sympatric species will cross

in the absence of a conspecific mate (Turner et al., 2001).

Additionally the effect of limited gene flow (e.g. from the

occasional migrant individual) may not significantly

affect overall development of reproductive isolation

(Tregenza, 2002). Large numbers of migrants would

likely only occur naturally through a major change in

connections between rivers, resulting in significant mix-

ing of populations.

Correspondence: J. S. Ready, Department of Vertebrate Zoology, Swedish

Museum of Natural History, P.O.Box 50007, 104 05 Stockholm, Sweden.

Tel.: 46 8 5195 4123; Fax: 46 8 5195 4212;

e-mail: jonathan.ready@nrm.se

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Keywords:

allopatric;

Amazon;

cichlid;

colour;

mate choice;

species.

Abstract

Laboratory mate choice experiments have confirmed species status for cichlid

fish in the African Great Lakes that differ in colour and little else. Colour

differences between allopatric populations of the South American cichlid

genus Apistogramma are known for many species, yet the status of such

populations has not been previously tested. Analysis of the genetic relation-

ships and mate choice characteristics of populations previously described as

Apistogramma caetei from eastern Amazonia indicates genetic differentiation

into at least three allopatric lineages, which also show strong prezygotic

isolation through female mate choice, confirming them as Biological species. If

future studies confirm that this result is indicative of a general trend, the

species richness of the South American cichlid fishes may presently be

seriously underestimated.

doi:10.1111/j.1420-9101.2006.01088.x

The designation of species status to colour morphs is a

contentious issue. However, the larger number of studies

involving colour delimitation for species of African

cichlids compared to South American cichlids is sug-

gestive of a greater understanding of the importance and

validity of such delimitation for this group of fishes. As

such, should it be shown that allopatric colour morphs

of South American cichlids are commonly cryptic spe-

cies, our estimates of their species richness are likely to

be far too low. This will have an impact on comparative

studies in evolutionary biology, where explanations for

variation in species richness of particular groups may be

sought.

Here, we aim to test whether the allopatric populations

of a South American cichlid represent biological species,

and to estimate the degree and duration of isolation and

the evolutionary history of these populations. We recog-

nize species must be practically diagnosable and use a test

for biological species as one method of delimiting species

among many.

Materials and methods

Experimental animals

The study was focussed on populations presently

assigned to the species Apistogramma caetei, Kullander

(1980), from rivers of eastern Amazonia. Female A. caetei

lay adhesive eggs onto a hard substrate, such as a rock or

piece of wood, usually in a concealed place within the

territory of a mature male (J. S. Ready, personal

observation). Females carry out most of the intimate

care of the progeny, but males usually remain in the area

and continue to defend their territory and offspring.

Males of cichlid fish species showing a similar mating

system have been recorded to show a degree of polygyny,

accepting a second mate within the same territory or

moving out to mate with another female (Ohm, 1958;

Burchard, 1965). Males are much larger than females,

with longer fins. Males show a subtle colour pattern of

iridescent blues, purples and reds, which varies between

species and populations regarded as conspecific (Koslow-

ski, 2002; Fig. 1 and supplementary material, Figs S1–

S3). By contrast, territorial and courting female Apisto-

gramma show a bold black and yellow colour pattern

which often varies little between populations and species

(Kullander, 1980; Koslowski, 2002).

Fish were collected from sample sites with location

recorded using GPS (Fig. 1). These included both live fish

for mate choice experiments (approximately 100 indi-

viduals from each of three sites in three river systems),

and as DNA samples with associated voucher specimens

(a minimum of 20 individuals from each of 16 sites in

five river systems). Apistogramma regani were obtained to

provide an outgroup for molecular analyses. Voucher

specimens were mostly deposited at the Universidade

Federal do Para (UFPa), the remaining submitted to the

collection of the Swedish Museum of Natural History

(NRM), with collection numbers (NRM52414–

NRM52421, NRM52423–NRM52425, NRM52427,

NRM52429–NRM52432, NRM52435–NRM52436 and

NRM52445).

Molecular methods

DNA extraction was performed at UFPa, campus de

Braganca, Brazil, using a standard phenol chloroform

extraction protocol. A fragment of the cytochrome b gene

was sequenced using the conserved primers L14841 and

H15149 developed by Kocher et al. (1989). The PCR

cycle: 1 · 94 �C for 5 min, 30 · (94 �C for 1 min, 48 �Cfor 1 min, 72 �C for 3 min), 1 · 72 �C for 10 min. The

mitochondrial control region was sequenced using the

primers A and E of Lee et al. (1995) with conditions as for

cytochrome b, except for an annealing temperature of

56 �C instead of 48 �C. Sequencing was performed on the

ALF system (Amersham Pharmacia Biotech, Freiburg,

Germany), using their own kit (Thermosequenase Primer

cycle sequencing Kit, Amersham) and recommended

thermal cycling parameters.

Analysis of molecular data

Sequences were aligned using BIOEDITBIOEDIT (Hall, 1999),

and nexus files prepared and checked with MACCLADEMACCLADE

(Maddison & Maddison, 2000). Control region sequences

show greater variability and phylogenetic signal, but the

cytochrome b gene fragment was chosen to perform

Nested Clade Analysis (NCA) in the package GEODISGEODIS

(Posada et al., 2000) as it did not contain indels. At least

ten individuals from each location were sequenced for the

cytochrome b fragment in line with sample numbers used

in previous studies using NCA (Templeton, 1998, 2001).

Cytochrome b fragment sequences are 346 bp long and

have the following GenBank accession numbers:

AJ557076–AJ557127, AJ297670 and AJ298941–

AJ298946. Control region sequences were from 361 to

367 bp in length. A small number of indels result in an

overall alignment of 370 bp. These have GenBank

accession numbers: AJ298951–AJ298978.

The program MODELTESTMODELTEST (Posada & Crandall, 1998)

was used to determine the best model for molecular

evolution in the samples. The AIC criteria selected the

model TVM + I + G model (I ¼ 0.717, G ¼ 0.4031) for

cytochrome b fragment analysis. The model selected for

the control region sequences was the HKY + G model

where G ¼ 0.3297. PAUP*PAUP* (Swofford, 1998) was used to

construct a heuristic parsimony tree (options: ACCTRAN,

TBR, MULTREES, Gaps ¼ missing, random addition,

ti/tv weighting and other characteristics from the models

selected by MODELTESTMODELTEST) using sequences of A. regani as

outgroup. Outgroup status was confirmed using other

Neotropical cichlid cytochrome b sequences from

GenBank. Sequences were then reanalysed with 1000

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bootstrap replicates under the same parsimony settings,

100 replicates using the heuristic search option for

maximum likelihood and 1000 bootstrap replicates for

neighbour joining. Bayesian support was obtained using

the program MRMODELTESTMRMODELTEST (Nylander, 2002) to deter-

mine the model and MRBAYESMRBAYES (Huelsenbeck & Ronquist,

2001). This involved 2 000 000 MCMC iterations, samp-

ling every 100 iterations with a 10% burn in (likelihood

values stabilized). A consensus of the remaining trees

was then obtained in PAUP*PAUP* (Swofford, 1998). Cyto-

chrome b sequences were used to produce a minimum

spanning network with the program TCS (Clement et al.,

2000) from which clades could be nested (summary data

online). From this an input file was constructed for

GEODISGEODIS (Posada et al., 2000), for NCA. The matrix

representing geographical relationships between sites,

which were compared with genetic distance was the

distance between sites through water (i.e. along the

rivers) as measured using scale maps and ARCVIEW 3.2ARCVIEW 3.2

(1992–1999). The information on geographical distances

is quantified into two main groups (Templeton, 1998):

the clade distance (Dc) for the geographical spread of a

clade; and nested clade distance (Dn) which measures

how a particular clade is geographically distributed

relative to its closest evolutionary sister clades (i.e. clades

in the same higher-level nesting category). Testing for

significantly small or large Dc or Dn distances in each

nested clade is then accomplished through Monte–Carlo

permutations (in GeoDis). A significant departure from

simulated randomness leads to rejection of the null

hypothesis of panmixia (no association between haplo-

type distributions and geography). For example, past

fragmentation events will tend to limit the geographical

range of clades. This results in significantly small clade

distances for both interior and tip clades with a signifi-

cant restriction in clade distances at higher clade levels

Fig. 1 Summary of the three colour forms

previously described as A. caetei and their

geographical locations. Marapanim males

show a purple colouration. Guama/Capim

males show a blue–grey colouration without

red cheek markings. Males from the other

Atlantic rivers show a blue–grey colouration

with red cheek markings and more iridescent

colour on the flank. Black – Marapanim;

Green – Quatipuru; Blue – Caete; Purple –

Piquioira; Orange – Jeju; Red – Apeu; Pink –

Capim; and Yellow – Guama.

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(Templeton et al., 1995). Other patterns for Dc and Dn are

expected for other population processes (Templeton

et al., 1995) and by following the most recent inference

key (available freely from the GeoDis website) it was

possible to determine which of these processes applied to

different clades.

In order to determine whether populations are more

recently or more anciently differentiated maximum and

minimum estimates for divergence times were obtained

using the nonparametric rate-smoothing algorithm

(NPRS) (Sanderson, 1997) in TREEEDITTREEEDIT (Rambaut &

Charleston, 2001). This method is appropriate when

evolutionary rates vary between lineages. To determine

whether evolutionary rates were variable among

lineages, the likelihood values of the overall topology

were calculated with and without a molecular clock

enforced in PAUP*PAUP* (Swofford, 1998) and subsequently

used to perform a likelihood ratio test (LRT). The test

statistic is Chi-squared distributed with n)2 d.f., where n

is the number of sequences (Muse & Weir, 1992).

Outgroups were removed prior to rate smoothing

calculations to prevent overestimation of the mean

evolutionary rate across the phylogeny. Branch lengths

were calculated for the constrained (most parsimonious)

tree using 100 bootstrap replicates and maximum like-

lihood settings. Branch lengths were then transformed

using NPRS as implemented in TREEEDITTREEEDIT (Rambaut &

Charleston, 2001). Standard error was then obtained in

PAUP*PAUP* (Swofford, 1998) by enforcing the molecular clock

and describing the trees using user-input branch lengths.

These are then converted to absolute ages using estimates

of divergence rate.

An estimate for cytochrome b divergence rate in

related fish is 1.16% per million years (Near et al.,

2003). However, variation may be quite large, and so

calculations were made using rates of both 1 and 2%.

Rates for the control region are higher, with a suggested

range of 3.6% ± 0.46% per million years (Donaldson &

Wilson, 1999). However, as it has been suggested that

particularly the 5¢ end of the control region could evolve

significantly faster (McMillan & Palumbi, 1997), so

divergence times were calculated for rates of both 3.14

and 7% per million years.

Mate choice experiments

Aquaria were set up in temperature maintained rooms

(24–28 �C) at the University of Hull, UK. Filtration was

provided by box filters run from compressed air supply.

Lighting was set on a 12 h on, 12 h off cycle. Live fish

were collected in 2001 and 2002 from the rivers Caete,

Quatipuru and Guama. Water chemistry was measured

to provide similar conditions in experimental aquaria (pH

6–6.5, <1 � GH and KH). Fish were exported to the UK by

Arapaima Ltd. in Belem, Brazil. Disease amongst fish was

monitored for and treated whenever required. Fish were

fed on a basic diet of flake and granules, with frozen food

(bloodworm, daphnia and brine shrimp) given occasion-

ally to encourage breeding condition. Most fish were kept

in large stock tanks (two for each population). Territorial

males were removed to experimental aquaria, or to male

isolation chambers in divided aquaria.

Mate choice was assessed in a tank 1 m long by 20 cm

deep and 30 cm front to back. Each half of the tank

contained a half coconut and flowerpot for use as a male

refuge and spawning cave. Males of similar size (<3 mm

difference in standard length of between 4 and 6 cm)

from different river systems were introduced and

monitored for aggression and territory formation. Hav-

ing similar-sized males prevents piracy (Mboko &

Kohda, 1999), size assortative mating by females

(Beeching & Hopp, 1999) and sneaker male mating, as

both males are clearly territorial adults (Makin, 1991).

Males could be identified by general colour pattern

associated with river system and/or individual markings

on the body and fins. When each male had established a

territory taking over approximately half of the tank, a

trial could be started. A mature female from the same

population as one of the males was then introduced.

Females were always selected from a different stock tank

to the same-population male in the trial to prevent

previous contact influencing the experiment. Even so,

imprinting on the population colour form may occur

(Barlow, 2000). This could only be avoided through

raising single fish in isolated chambers, which was

prohibitive for this study. Cross fostering would not be a

practical solution in this case as fry would be eaten

rather than adopted. If accepted by both males, the

female was allowed to remain in the tank until she

spawned. Trials were stopped if one male lost his

territory or if the female was being aggressively chased

by either male. Spawning usually occurred within

3 days to 1 week. Choice of male was assessed by

recording the identity of the territorial male who

controlled the spawning cave in which eggs were seen.

Different individual fish were used in each experiment

and replicate comparisons for each combination of males

were carried out in different aquaria to control for any

imperceptible differences between aquaria.

Results

The general topology of the haplotype network (Fig. 2a)

indicated distinct geographical structuring with a large

number of missing haplotypes separating the Marapanim

population from all others. Following the latest inference

key, NCA confirmed the fragmentation of the popula-

tions from the Marapanim and other Atlantic draining

rivers and also indicates either ‘long distance coloniza-

tion’ or ‘past fragmentation followed by range expansion’

as the history relating populations of the Guama and the

remaining Atlantic draining rivers (highlighted in Fig. 3).

Additionally, the haplotypes from the Caete, Quatipuru

and Piquioira are inferred to show allopatric fragmenta-

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tion. The mitochondrial control region indicates phylo-

genetically distinct groupings of fish from three areas: the

Marapanim, Guama and the Caete/Quatipuru, with

some indication that there may be some difference even

between the Caete and Quatipuru populations (Fig. 2b).

In summary, in the Bragantina region of Brazil, there

appears to be at least three diagnosable genetic groups of

Apistogramma occupying the different river systems. The

maximum and minimum divergence time estimates

including standard error suggest that the Marapanim

lineage has been diverging from the other Atlantic

drainages for between 1.37 and 0.17 million years

(4.4% mean pair-wise divergence in cytochrome b), that

the other Atlantic coastal drainages have been diverging

from the Guama/Capim drainage for between 1.11 and

0.12 million years (3.7% mean pair-wise divergence in

cytochrome b), and the Guama/Capim drainage has been

diverging from the Marapanim for between 1.52 and

0.28 million years (4.7% mean pair-wise divergence in

cytochrome b), while the maximum divergence within

these groups is between 0.5 and 0.05 million years (1.6%

maximum pair-wise divergence in cytochrome b).

(a)

(b)

Fig. 2 Genetic differentiation of populations

previously described as A. caetei. Colours

correspond to those in Fig. 1. (a) Network of

44 partial sequence cytochrome b hap1o-

types from 180 individuals. Circular area

represents number of sequences and white

squares represent missing intermediate

haplotypes. (b) ML phylogram of 27 partial

mitochondrial control region sequences.

Outgroup A. regani removed to better view

distances and support. Support from parsi-

mony [1000 bootstrap, above left (CI ¼0.8831, RI ¼ 0.9698, RC ¼ 0.8564)], Baye-

sian posterior support (above right), Neigh-

bour joining (1000 bootstrap, below left) and

maximum likelihood (100 bootstrap, below

right).

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Haplotypes 1-step clades 2-step clades 3-step clades 4-step clades

No. Dc Dn No. Dc Dn No. Dc Dn No. Dc Dn No. Dc Dn

1 0 0

2 0 0 1-1 0S 151L

4 0 192

5 0 192

I-T 0 0 1-2 192 105

3 51.7S 68.7S1-3 81.8S 89.6S

6 0S 170LI-T 4.9 -42S

2-1 97.8S 101S

1-2-11-12-13-14 No: RE 1-2-11-12-13-14 No: RE

7 0 0 1-4 0 0

9 0 0 1-5 0 0 2-2 0 138

8 0 0 1-7 0 0 2-3 0 136

I-T -95 35.5L 3-1 106 105L

1-2-3-4 No: IBD

10 0 0 1-9 0 0 2-4 0 0 3-2 0S 92

11 0 0 1-10 0 0 2-5 0 0 I-T -106S -13.2 4-1 104S 321L

12 0 0 1-19-20-2-11-12 No:

CRE

16 0 0 1-16 0 0 2-7 0 0

17 0 0 1-14 0 0

18 0 0

19 0 0 1-15 0 0 2-8 0 0

20 0 0 1-17 0 0

21 0 0

22 0 0 1-18 0 0 2-9 0 0 3-3 0S 374L

13 0 0

14 0 0

15 0 0 1-12 0 0 2-6 0S 453L

23 0 0 1-20 6.4S 87.5S

28 0 0 1-21 0S 157L

24 0S 45.3

25 0S 41.6

26 0S 58.8L

27 0 50.7

I-T 0 -4.5S1-22 49S 107

1-2-11-12-13-14 No: RE

29 11 10.9 1-23 10.9S 95S

30 0 7.6 I-T -22S -29S2-10 111S 135S 3-4 155S 194S

I-T 11 3.3 1-19 No: AF 1-2-11-12-13 Yes: LDC

or PF followed by RE

I-T 155L -180S 4-2 220S 288S

1-19-20 No: IGS I-T 116L -33S

1-2-3-5-6: Too few. RE/C

or RD/GF

31 0 0 1-24 0 0

32 0 0

34 0 0 1-25 0 0

33 0 0 1-26 0 0 2-11 0S 15.3

42 0 0 1-30 0 0

43 0 0 1-31 0 0 2-13 0 20.7

35 20 17.9

36 0 12.5

I-T 20 5.4 1-27 18.8 19.2S

40 28.3 26.8 1-29 27.4 25.2L

Linking the clade groups:

1-19 No AF (has many

mutational steps)

41 0 15.3

38 0 0 1-28 0S 22.6

39 0 0

37 0 0 1-32 0 17.5S

44 0 0 I-T 19.6L 5.4L2-12 24.5L 22.9L

1-2-3-4 No: IBD I-T 21 1.1 3-5 1-2-3-5-6: Too few. RE/C

or RD/GF

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Mate choice tests (raw data in online material)

revealed significant assortative mating (Table 1), with

females generally only choosing to mate with males from

the same population. The exception was an instance

where a female of the Quatipuru population mated with

a male from the Caete population, resulting in overall

partial assortative mating between these populations –

the most genetically similar of those in the trial. The F1offspring maintained the colour differences of each

population despite being raised in aquaria, indicating a

genetic basis for the colour differences.

Discussion

Genetic divergence between the populations of Apisto-

gramma in the Bragantina region was not very large by

many standards, though it is much larger than diver-

gences between many species and even genera of cichlids

from the African Great Lakes (Lee et al., 1995; Turner

et al., 2001). Significant genetic divergence of fish from

the Marapanim drainage was evident when analysing

both control region and cytochrome b sequence data

(Figs 2 and 3), while significant divergence between fish

from the Guama/Capim drainage and those from the

Caete, Quatipuru and Piquioira was fully supported by

analysis of control region sequence data and partially

supported as one of two possible historical inferences by

the analysis of clades 3 and 4 (comprizing clades 2–6 and

2–10) of the nested haplotype network of cytochrome

b fragment sequences. Finally, there is some evidence for

genetic divergence between the populations from the

Caete, Quatipuru and Piquioira, though with a short

branch length for the phylogram produced from control

region sequences and an inference of allopatric fragmen-

tation (rather than the more definite inference of past

fragmentation) from NCA. The weaker support for the

latter situations suggests a series of splits in the recent

history of these fish either as a result of changing patterns

in the drainage systems they occupy or as a result of

isolation after colonisation. Although the divergences

outlined above are relatively small their strength as

indicators of biological units is confirmed by the results of

the assortative mating trials. Estimates of divergence time

between reproductively isolated populations are relat-

ively small, supporting recent, rather than ancient,

divergence of the populations studied. The maximum

estimates for divergence between these populations of

around 1–1.5 million years ago are within the normal

estimates of rates of allopatric speciation in other taxa

(Coyne & Orr, 2004).

Mate choice trials indicated a high level of assortative

mating between populations, consistent with their status

as biological species, or at least, incipient species. Our

experiments were carried out with fish taken from the

wild and kept in stock tanks with conspecifics. Thus, our

experimental design cannot allow us to dismiss the

possibility that preference for mating with individuals of

the same population may have a learned component.

However, there is very little evidence for a major effect of

sexual imprinting in cichlid fish. Cross-fostering experi-

ments with heterospecifics and experimental tests of

mate preferences of colour polymorphic species reared

with parents of different colour have indicated very weak

effects on behaviour and no alternation in mate prefer-

ence (Barlow, 2000). With the number of mate choice

trials performed, it was not possible to determine the

overall effect of partial assortative mating between Caete

and Quatipuru populations. However, evolution of

reproductive isolation does not require either complete

Fig. 3 Results of the nested geographical analysis of A. caetei cytochrome b fragment haplotypes as determined using the NCA inference key.

The nesting was based on the haplotype network in Fig. 2a. Haplotypes, number of sequences and corresponding colour in Figs 1 and 2 are

given in Table S1. Following the name or number of any given clade is the clade (Dc) and nested clade (Dn) distances. Also in those nesting

clades containing both tip and interior nested clades, the average difference between interior vs. tip clades for both distance measures is given in

the row labelled I-T. A superscript S means that the distance measure was significantly small at the 5% level, and a superscript L means that the

distance measure was significantly large. At the bottom of the boxes that indicate a set of clades in which one or more of the distance measures

was significantly large or small is a line indicating the biological inference as determined by the inference key. The numbers refer to the

sequence of questions in the key that the pattern generated, followed by the answer to the final question in the key. Following this answer is

the biological inference generated by the inference key, where RE is range expansion, AF is allopatric fragmentation, IBD is recurrent gene flow

restricted by isolation by distance, LDC is long-distance colonisation, PF is past fragmentation, RE/C is range expansion/colonisation, RD/GF is

restricted dispersal/gene flow, CRE is contiguous range expansion and IGS is inadequate geographic sampling. If two or more of these symbols

are after the colon, the inference key could not distinguish among the indicated alternatives. Grey highlighting indicates support for

fragmentation of populations in different drainages.

Table 1 one-tailed binomial statistical test for deviation from

random mating between populations previously described as

A. caetei. Choice of either male by the female has P ¼ 0.5 under the

null hypothesis of random mating.

Female

Other male

in test

Same

population

matings

Other

population

matings P (*significant)

Caete Quatipuru 6 0 0.015625*

Caete Guama 6 0 0.015625*

Quatipuru Caete 5 1 0.109375

Quatipuru Guama 6 0 0.015625*

Guama Caete 6 0 0.015625*

Guama Quatipuru 6 0 0.015625*

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assortative mating nor complete hybrid inviability or

infertility to occur, and correlations between phenotypic

divergence and genetic divergence may be weak

(Tregenza, 2002). Additionally, post-zygotic isolation is

generally associated with divergence time between spe-

cies, and often occurs after speciation if prezygotic

isolation is also acting (Coyne & Orr, 1999; Price &

Bouvier, 2002). Although colour may not allow distinc-

tion of the Caete and Quatipuru populations it is possible

that these populations could recognize each other as

different based on other cues. Sound (Amorim et al.,

2004) and olfactory cues (Plenderleith et al., 2005) have

recently been implicated in species recognition in closely

related species of African cichlids. As such, the assortative

mating and genetic divergence found between the

populations of Apistogramma from the Bragantina region

combine to fully support at least three distinct species

which can be identified on the basis of colour differences,

one from the Marapanim, one from the Guama/Capim

drainage and one from the Caete, Quatipuru and

Piquioira drainages. As we could not find means of

diagnosing fish from the Caete from those of the

Quatipuru, species delimitation is not yet practical for

these populations. Although assortative mating experi-

ments were not performed for the Marapanim popula-

tion, these fish are even more genetically divergent from

the populations which do mate assortatively and exhibit

colour differences, thus supporting their species status.

The potential for partial assortative mating or reduced

fitness of offspring to lead to incipient or actual speciation

increases when gene flow is limited. This is likely the case

in Apistogramma species where dispersal is incredibly low

(Walker et al., 1991) and, considering the overall partial

assortative mating between Apistogramma from the Caete

andQuatipuru, supports the possibility ofmore than three

species being present in the study area. However, as no

phenotypic differences could be observed between the

Caete and Quatipuru samples, diagnosis, and therefore,

delimitation, as different species is not possible. If the

Caete and Quatipuru populations have not already spe-

ciated, they are probably currently undergoing speciation.

It seems likely that Apistogramma species in eastern

Amazonia have diverged recently in allopatry via selec-

tion and/or drift to form biological species. We recognize

three species within what was previously described as

A. caetei. All three species have the morphological

characteristics of A. caetei as described by Kullander

(1980). Apistogramma sp. 1 from the Caete, Quatipuru

and Piquioira rivers is distinguished by the presence of

red markings on the cheek and iridescent blue markings

on the flanks of males. Males of Apistogramma sp. 2 from

the Guama river have dull grey colouration (no irides-

cent marks on the flanks) and lack red pigment on the

cheeks. Male Apistogramma sp. 3 from the Marapanim

river drainage has purple colouration particularly asso-

ciated with certain fins and the operculum. They also

lack red pigment on the cheeks.

The pattern of molecular and morphological diver-

gence found in cichlids is typical of that found for inland

waters generally (Hebert, 1998). Groups of species from

deep, old lakes (including some East African haplochro-

mines) often show little genetic divergence, but striking

morphological change, while groups of species from

other aquatic environments (including Apistogramma)

show genetic structuring, but little morphological vari-

ation.

Although much is made of the terrestrial biodiversity

of the Neotropics and Amazonia in general, the aquatic

diversity is astounding. It has recently been estimated

that 6000 of the world’s 13 000 freshwater fish species

(46%) are found in South and Central America (Reis

et al., 2003). If the pattern here is repeated in central

Amazonian Apistogramma species, then the number of

species in the genus is likely to be significantly higher

than currently thought. From the current number of

just over 60 valid species of Apistogramma we expect the

species richness of the genus to rise. A lower estimate

of 120 species can be made based on 30–40 unnamed

species and the division of colour forms from three

wide ranging species with numerous colour morphs

(S. O. Kullander, personal communication), while a

maximal estimate of around 400 species assumes 30–40

unnamed species, 20–30 as yet undiscovered species

from remote areas, and that the pattern of isolation

found in this study is common to Apistogramma from all

clear and blackwater habitats leading to a trebling or

quadrupling of the sum of described and undescribed

species.

Acknowledgments

Work was supported by a PhD studentship from the

Fisheries Society of the British Isles awarded to JSR. We

also thank Domino Joyce, Cock van Oosterhout, Ole

Seehausen, Ola Svensson and Rafael Zardoya for discus-

sion and review and two anonymous referees for review

of the manuscript.

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Supplementary Material

The following supplementary material is available for this

article online:

Table S1. Numbers of sequences for each cytochrome

b fragment haplotype and corresponding population and

colour used in Figs 1–3.

Table S2. Raw data from mate choice trials.

Fig. S1. Adult male Apistogramma sp. 1 from the Caete,

Quatipuru and Piquioira river systems.

Fig. S2. Adult male Apistogramma sp. 2 from the Guama

river system.

Fig. S3. Adult male Apistogramma sp. 1 from the Mara-

panim river system.

This material is available as part of the online article

from http://www.blackwell-synergy.com

Received 1 September 2005; revised 9 January 2006; accepted 9 January

2006

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