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Assessment of the use of DNA barcoding for identification of some

By

OLUSHOLA AHMED OLALEYE

DISSERTATION

Submitted in fulfilment of the requirements for the degree

MASTER IN ZOOLOGY

at the

UNIVERSITY OF JOHANNESBURG

May 2017

SUPERVISOR: PROF HERMAN VAN DER BANK

CO-SUPERVISOR: PROF JOHN MAINA

DECLARATION

I declare that this dissertation hereby submitted to the University of Johannesburg for the degree MAGISTER SCIENTIAE (Zoology) has not been previously submitted by me for the degree at this or any other University, that it is my work in design and execution, and that everybody who contributed and all materials contained therein has been duly acknowledged.

OLUSHOLA AHMED OLALEYE May 2017

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ACKNOWLEDGEMENT

My deepest gratitude goes to Prof. Herman van der Bank and Prof. John Maina for their supervision. Special thanks to Dr Olivier Maurin, Dr Kowiyou Yossofou and Mr

Ronny Kabongo for their support and assistance in making the analyses of this study possible. Also, my sincere gratitude to Pavlinka for her immense and swift support in providing articles and textbooks that where difficult for me to get.

My heartfelt appreciation to Prof. Mitchelle van der Bank and Faculty of Science

University of Johannesburg for provision of funding for this project.

A big thank you my dear colleagues and friends: Mariam Adeoba, Anifat Bello, Henry

Ihenachio, Ojelade Solomon, Oloruntoba Bamigboye, Oluwatobi Bamigboye,

Olakunle Oyekanmi, Akinseye Samuel, Lindi Steyn and Dr Bezeng S. Bezeng for their assistance, suggestions and corrections in the different parts of this study.

I appreciatively acknowledge Dr Lolo Mokae, Dr Ololade Shonubi, Dr Seyi Olokede and Dr Tola Odubajo for their enormous support, advices and time throughout the study.

I thank the African centre for DNA Barcoding for its infrastructural support for the duration of this project.

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To my ever-supportive family, my dad Alhaji Azeez Olaleye, my mom Elizabeth

Olaleye, my sister and her family the Ogunbinu-Peters and my brother and his family the Olaleyes; I gratefully appreciate and thank you for believing in me and always encouraging me in all my endeavors.

Also to my lovely wife Palesa, my daughter Matshepo and son Olusegun for being my greatest motivation for accomplishing this study. I truly appreciate all your support, prayers and encouragement my wife.

Lastly and above all appreciations, I thank God Almighty for bestowing strength,

Mercies and grace through all the duration of this study. Oluwa mi e seun gan. Oluwa mi modupe.

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ABSTRACT

Species identification and delineation are very important in studies such as biodiversity, conservation biology, ecology and evolutionary biology. are the common model group of which are used to explicate the mechanisms of adaptation, speciation and diversification. Application of combined molecular genes (such as 16S, Cytochrome oxidase subunit I (COI), histone H3 & recombination activating gene two TMO4C4) on the phylogeny of cichlids has played an important role in cichlid phylogenetic studies. Here, identification of cichlids were assessed using DNA barcoding and phylogenetic tree reconstruction. All analyses were based on a single mitochondrial gene COI. Sequences of grahami and albolabris were generated for the first time along with mined sequences from Barcode Of Life Data base systems (BOLDsystems). BRONX (Barcode Recognition Obtained with Nucleotide eXposés) was also used to confirm unique sequences representing each species. The performance of COI gene was tested based on three identification metrics namely best close match (bcm), near neighbour (nn) and BOLD identification criteria (BOLD id) using SPIDER in R. Next, occurrence of barcode gap was established on the difference of furthest intraspecific distance among its own species and the closest, non-conspecific distance (nonConDist – maxInDist). A maximum parsimony and a Bayesian inference (bi) tree were reconstructed to further investigate species delimitation among cichlid fishes. Both nn and bcm showed 89% true and 87% correct identification respectively in the dataset whereas BOLD id gave a lower correct identification (54%). There were well-supported values at each node of bi tree reconstruction, indicating evolutionary divergence among cichlids. COI gene performed well in the identification of Alcolapia and Thoracochromis (nn and bcm = 100%). It also performed well in the phylogenetic positions of Alcolapia and Thoracochromis (support values of 92% and 100% respectively). However, populations of Alcolapia grahami were not fully resolved by COI gene. The COI barcoding gene is viewed as an efficient tool for enhancing species identification and delimitation. Morphological data and nuclear genes or microsatellites should be added to resolve the ambiguity in the problematic specimens.

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TABLE OF CONTENT

Declaration…………………………………………………………………………..… i

Acknowlwdegments…………………………………………………………………. ii

Abstract………………………………………………………………………..………. iv

List of figures…………………………………………………………………………. viii

List of tables…………………………………………………………………………... ix

1. Introduction……………………………………………...………………..………… 1

1.1 General introduction to cichlids.………………………………………………… 1

1.2 Cichlids of economic importance……………………………………………...... 2

1.3 Distribution of cichlids……………………………………………………………... 3

1.4 Biogeography of cichlids………………………………………………………….. 6

1.5 Lacustrine cichlids…………………..……………………….……….. 9

1.5.1 Brief biogeography of some cichlids……………… 9

1.5.2 Brief biogeography of some cichlids…………………… 10

1.5.3 Brief biogeography of some cichlids…………………… 10

1.5.4 Brief biogeography of Lake Magadi cichlid …………………………… 11

1.6 Riverine Old World cichlids……….……………………………………………..... 13

1.6.1 River Congo cichlid distribution ………………………………………... 13

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1.6.2 Cichlid fishes of the southern African freshwater systems…………... 14

1.7 Phylogenetic relationships among cichlid assemblages………………………… 15

1.8 DNA barcoding – a molecular tool for species identification………………….… 18

1.9 Hypotheses and Objectives of the study………………………………………… 23

2. Materials and methods…...………………………………………………………….. 23

2.1 Locations and habitat of species sampled……………………………………….. 25

2.2 Sampling of taxa…………………………………………………………………… 25

2.3 DNA extraction……………………………………………………………………… 30

2.4 PCR amplification…………………………………………………………………… 32

2.5 PCR reaction clean-up……………………………………………………………… 33

2.6 DNA cycle sequencing……………………………………………………………… 34

2.7 DNA sequence analysis…………………………………………………………… 34

2.8 DNA Barcoding……………………………………………………………………… 34

2.9 Phylogenetic tree reconstruction…………………………………………………… 36

3. Results...……………………………………………………………………………….. 38

3.1 Extraction of DNA and amplification of COI gene……………………………….. 38

3.2 Sequence mining and filtering……………………………………………………… 40

3.3 DNA barcoding……………………………………………………………………… 40

3.4 Outline of nucleotide variation….……………………………………………….… 50

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3.5 Phylogenetic relationship among Cichlidae…...………………………………… 51

3.6 Relationship among some species within the lineage.………… 57

4. Discussion…………………………………………………………...………………… 59

4.1 Phylogenetic relationships…………..……………………………………………… 61

5. Conclusion………………………………………………………...…………………… 66

References……………………………………………………………………………..… 68

Appendix………………………………………………………………………………….. 91

1. Cichlid authorities………………………………………………………………… 91

2. The distribution of cichlids used in this study..………………………………... 94

3. The position of base pair where variations occurred within the two populations of Alcolapia grahami sampled from Springs Lagoon (FSL) and South West Lagoon (SWL)……………………………………………………………...100

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LIST OF FIGURES

Figure 1. Distribution of the family Cichlidae showing some barcoded locations

(squares) of cichlid species in the world (www.boldsystems.org) ...... 5

Figure 2. Map of indicating the distribution of some African cichlids ..…...... 27

Figure 3. Distribution of Magadi (Alcolapia grahami).………………..………. 28

Figure 4. Distribution of Thoracochromis in ………………. 29

Figure 5: Gel electrophoresis products of clean DNA of two African cichlid fishes viewed in 1.5% agarose gel with the aid of ultra-violet light…………………….…... 38

Figure 6: Gel electrophoresis products of PCR products and clean PCR products…………………………………………………………………………..………. 39

Figure 7: Barcode gap evaluation of cichlid family represented in this dataset…… 46

Figure 8. Maximum parsimony tree based on mtDNA COI data of some cichlids………………………………………………………………………………….…. 52

Figure 9. One of the 73 most parsimonious trees based on mtDNA COI data of representative of Oreochromini lineage…………………………………………...….. 56

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LIST OF TABLES

Table 1: Barcoding summary on the genera with at least four sequences………… 43

Table 2: Percentage barcode gap in entire dataset applying the Meier et al. (2006) method……………………………………………………………………………………. 47

Table 3: Identification efficacy of DNA barcodes applying distance based methods in percentage……………………………………………………………………………….. 49

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1. INTRODUCTION

1.1 General introduction to cichlids

The most diverse group of extant vertebrates on earth are fishes (e.g. Nelson 2006).

According to Nelson (2006), over 24 600 living fish species are known. They occupy aquatic environments such as rivers, lakes, estuaries, seas, and oceans. The cichlids are one of the largest group of fish (e.g. Kocher 2004). Cichlids belong to the order

Perciformes in the infraclass Teleostei (bony fishes) and in the class

(ray-finned fishes) (Nelson 2006). Members within the cichlid family include both brackish and mostly freshwater fishes (Dunz 2012).

The size of the cichlid family, the complexity of environmental interactions and the fast evolutionary development have all contributed to the speciation of this family (Berra

2007). In addition, cichlids exhibit remarkable social and morphological variations following convergent speciation (Berra 2007).

Cichlid fishes display certain distinctive and strong parental care as well as various breeding methods such as monogamy, polygyny, polyandry and polygynandry (Barlow

1991; Sefc 2011). Based on their reproductive behavioural patterns, generally, cichlids are divided into two groups: i) mouth brooders (where eggs and larvae are protected in the mouth) and ii) substrate brooders (where eggs and fry are protected in the nest)

(Barlow 1991; Sefc 2011).

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The type of food that is found in the ecological niche of cichlids has contributed to the modification of the functional morphology of their feeding apparatus (Liem 1991). From their various forms of feeding apparatuses, the cichlids have been further categorised according to their feeding patterns into grazers (e.g. spp.), browsers

(e.g. spp.), planktivores (e.g. spp.), piscivores (e.g.

Lepidiolamprologus spp.), insectivores (e.g. spp.), specialist algal scrappers (e.g. spp), paedophages (e.g. Lipochromis spp.), molluscivores

(e.g. spp.) as well as lepidophages (e.g. spp.) (Yamaoka

1991; Stauffer and Madsen 2012).

The physical appearance of cichlids can be fascinating to aquarists and aquaculturists because they have vast array of colour, shapes and sizes (Berra 2007). Majority of these fishes are of great economic importance worldwide.

1.2 Cichlids of economic importance

There are many cichlid genera like , Cichlosoma, Pterophyllum,

Hemichromis etc., which are used in aquaculture because of their ease of farming

(Pullin 1991). Many members of the family serve as an important food source (Skelton

2001) for humans as well as for some aquatic . For example, the are among the most consumed fishes globally (Modadugu and Belen 2004). One of the cichlid species, niloticus is ranked second to Carp spp. as the most farmed fish in the world (Jegede 2011). Some cichlids are ornamental aquarium specimens (Skelton 2001) e.g. ocellatus, nigrofasciatum,

Thorichthys meeki etc. (Raja et al. 2014). In some of the world e.g. ,

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Cichla are well-known in angling sport (Greenwood and Stiassny 2002). Additionally, cichlids have been reported to be of medical importance (Eddyani et al. 2004). Some cichlid fishes such as Hemichromis bimaculatus, H. fasciatus, O. niloticus, and Tilapia guineensis are known to transmit Mycobacterium ulcerans, the cause of Buruli ulcer

(Eddyani et al. 2004). This is because being microphagous, they possess gill systems with very fine network, capable of entrapping tiny particles such as bacteria (Gosse

1965). Species such as Metriaclima lanisticola and are very effective agents for biological control of schistosomiasis (disease transmitted by parasitic helminths, the Schistosome spp.) (Lundeba et al. 2007; Stauffer and Madsen

2012). These two species are molluscivorous fishes that prey on the intermediate host

(snails of the Bulinus sp.) of the parasite (Stauffer and Madsen 2012).

1.3 Distribution of cichlids

Most cichlid fishes can be found in regions where climatic conditions are humid and hot (Greenwood and Stiassny 2002). In general, cichlid fishes are distributed throughout tropical waters around the earth. In particular, Cichlids spread across mainland Africa, the Neotropics (Central and ), and India

(Farias et al. 1999; Turner et al. 2001) (Fig. 1), with their biodiversity hotspot being in

East Africa (Salzburger and Meyer 2004). Over the years, the exact numbers of cichlid species in Africa or globally have been assumed to be between 2 000 and 3 000 by different Ichthyologists (e.g. Snoeks 2000; Koblmüller et al. 2008). This is due to the high morphological variability in the group to an extent that even within a single species, some populations are morphologically different (Turner 2007). Some species e.g. angustiuceps lack characteristic morphological features

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especially during their developmental stages (Van der Bank et al. 2009). Such difficulty in morphological identification hampers the ability of fully estimating the total number of cichlid species that are found in Africa in particular and on earth in general. Recently,

1 677 well-described species of cichlids were identified globally (Eschmeyer and Fong

2015): this was an increase from 1 600 species previously identified by Turner (2007).

Emanating from the vast number of species and rapid speciation, studies on origin and biogeographical distribution of the cichlids have been done (e.g. Vences et al.

2001; Sparks and Smith 2004; Musilová et al. 2008). A list of the authorities that described some of the cichlid species mentioned in this study is given on Appendix 1.

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Fig. 1. Distribution of the family Cichlidae showing some barcoded locations (squares) of cichlid species in the world (www.boldsystems.org).

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1.4 Biogeography of cichlids

The importance of cichlid fishes in evolutionary research and their biogeographic history has remained challenging (Vences et al. 2001; Genner et al. 2007; Azuma et al. 2008;

López-Fernández et al. 2013). This is mainly due to the extensive distribution of cichlid fishes all over the world. The biogeography of cichlid fishes was influenced by the split of

Gondwana which took place at the Early Cretaceous (Sparks and Smith 2004; Murray

2001). The effect of this supercontinental splitting caused the cichlids to disperse widely in the tropical areas of the Old World and subtropical areas of the regions

(Farias et al. 2000; Stauffer and Gray 2004; Musilová 2011). According to Farias et al.

(2000), cichlid lineages of the New World group are those from southern subtropical regions of Central and South America. This is because the South American separated from the remaining Gondwana landmass during the Early Cretaceous era about 99-145 million years ago (Friedman et al. 2013). On the other hand, lineages of the

Old World are those from mainland and Madagascar (Farias et al. 2000;

Stauffer and Gray 2004). The African lineages have the most remarkable patterns of radiation and speciation. Based on molecular study, Malagasy cichlids were placed at the root of all cichlid fishes (Farias et al. 2001). According to Sparks (2008), there are two lineages of the Malagasy cichlids: one lineage is exclusive to Madagascar and the other is common to the lineages from the South . Madagascar is thought to be the origin of the cichlids (Musilová 2011). Views about the biogeographical dispersal of cichlids have so far been controversial (Hay et al. 1999; Wainwright et al. 2012; Friedman et al. 2013).

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Based on their mode of distribution (Dunz 2012), the evolution and biogeographical history of the cichlids have been speculated on. One of such speculations is the drift vicariance which occurs when geographical barrier splits a particular population or the entire biota, leading to species fragmentation. This event supports Gondwanian distribution (Dunz 2012). Over the past decades, a vicariant event has been attributed to cichlids distribution, indicating the old age of the Family (Stiassny 1991; Zardoya et al.

1996; Farias et al. 1999). The discovery of Mahengochromis that is the oldest fossil of the cichlids is dated as far back to the middle Paleogene about 41 million years ago (Murray

2000a, b). However, according to Murray (2001) and Friedman et al. (2013), the least possible anticipated age of cichlid evolution was during the early Paleogene era (about

65 million year ago). This is in discordance with the findings of Hay et al. (1999) on the

Godwanan break-up that occurred during the early Cretaceous (about 99-145 million year ago). The theory of drift vicariance for the Gondwana dispersal is backed by the monophyletic sister groups on all Gondwana landmasses as indicated by many phylogenies of cichlids (Schliewen and Stiassny 2003; Sparks and Smith 2004). The theory of drift vicariance also favours the ability of some cichlids such as Alcolapia grahami to have subsisted in greatly alkaline water of Lake Magadi (Kenya) (Wilson et al.

2000). In addition, there was evidence of the vicariant event revealing the sister taxa

(Pholidichthyidae) of cichlidae to have originated from the marine environment

(Wainwright et al. 2012). This finding is in concordance with Dunz (2012) that there was dispersal of cichlids across the marine environment.

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Cichlid fishes of the African lakes provide unique opportunities of investigating the evolutionary processes (Ricklefs and Schluter 1993; Farias et al. 2000). Some adaptations of cichlids relative to body shape, colour patterns, behavior, enormous diversity and ecological modifications have made them remarkable creatures to study

(Kornfield and Smith 2000; Kocher 2004; Salzburger and Meyer 2004). In , lacustrine cichlids (cichlids found in Lake Magadi and mostly in Lakes Tanganyika, Malawi and Victoria) have been studied more compared to other cichlids in the world (e.g.

Hoerner 2011; Joyce et al. 2011). Cichlids have shown a capability of rapid speciation with their enormous diversity in three Great East African Lakes (Turner et al. 2001; Kocher

2004; Turner 2007). This ability has made them a model system for understanding their adaptive radiation, speciation and developmental biology (Friedman et al. 2013).

According to Salzburger et al. (2005), some key innovations leading to the Lacustrine Old

World cichlids evolutionary success, amidst others, are established on diverse ecological chances (establishment of colonies in large lakes), morphological characters (e.g. egg- spots, colour polymorphisms, pronounced sexual dichromatism) and reproductive behavioural traits (e.g. maternal mouth brooding). Below is a brief discussion of some Old

World cichlid’s biogeographical distributions.

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1.5 Lacustrine Old World Cichlids

1.5.1 Brief biogeography of some Lake Tanganyika cichlids

Lake Tanganyika has been in existence since the middle Cenozoic (about 9-12 Million years ago) (Danley et al. 2012). This makes it the oldest of the Great Lakes (Lake Malawi and Lake Victoria) in Africa (Danley et al. 2012). It is the longest (650 kilometers) and deepest (depth of 1470 meters) lake in Africa (Danley et al. 2012). With approxiamtely

250 species of cichlids (Danley et al. 2012), cichlids represent the most diverse group among the freshwater fish found in Lake Tanganyika (Salzburger et al. 2002). These species count up to 49 genera which are further grouped into 14 tribes (Takahashi 2003;

Salzburger et al. 2005). In addition, within these 14 tribes, some riverine ancestral lineages have possible distribution in this group (Salzburger et al. 2002, 2005). Some cichlids found in Lake Tanganyika show a typical example of intralacustrine allopatric speciation (Fryer and Iles 1972). However, Neolamprolagus brevis and

Boulengerochromis microlepis with 3cm and 80cm long respectively are one of the most prevalent species of cichlids found in Lake Tanganyika (Salzburger et al. 2002).

Salzburger et al. (2005) reported that Lake Tanganyika could plausibly be the origin of the Haplocromine ancestor of the species flock of Lake Malawi and Victoria.

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1.5.2 Brief biogeography of some Lake Malawi cichlids

Lake Malawi is about three million years old (Meyer et al. 1990), with depth of about 700 metres (Danley et al. 2012). Cichlid evolution occurred about 700 000 years ago in Lake

Malawi (Meyer et al. 1990). Approximately 800 species belonging to the Lake Malawi cichlid flock were identified by Konings (2007). These species are sub-grouped into two lineages namely the and Tilapiines (Turner et al. 2001; Danley et al.

2012). These two lineages are of a non-monophyletic origin (Joyce et al. 2011; Genner and Turner 2012) although the tribe within the Haplochromines lineage is monophyletic (Reinthal 1987). The Mbuna is dominated by the lithophilus (stone loving) genera including Genyochromis, etc. The study of Ribbink (1991) infers that lithophilus cichlids of Lake Malawi might be the ancestor of cichlids of the Great Lakes based on their geographical distribution pattern. There has been convergent evolution both in trophic morphologies and in colour between the Lake Tanganyika and Lake

Malawi cichlid fishes (Kocher et al. 1993; Muschick et al. 2012).

1.5.3 Brief biogeography of some Lake Victoria cichlids

With an estimated age of 800 000 years (Danley et al. 2012), Lake Victoria appears to be the youngest of the three Great Lakes (Meyer et al. 1994). On earth, with surface area of

68 800 sq kilometres and a maximum depth of 79 metres, the Lake is the widest tropical lake (Danley et al. 2012). Lake Victoria contains approximately 700 species (Turner et al. 2001). According to Verheyen et al. (2003), the cichlid species flock of Lake Victoria is diphyletic, i.e. they may have evolved from two separate lineages.

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Findings based on the molecular phylogeny of the cichlid fishes of Lake Victoria showed that the chief lineage evolution of this fauna occurred approximately 100 000 years ago

(Verheyen et al. 2003). Usually, Lake Victoria species flock is referred to as a “super- species-flock” (Meyer et al. 1994). This is because they are closely associated with the less species-rich groups occurring in the surrounding lakes such as Lakes Edward,

George and Kivu (Verheyen et al. 2003; Salzburger and Meyer 2004).

1.5.4 Brief biogeography of Lake Magadi cichlid

The Lake Magadi which is one of the most alkaline lakes in Africa (Talling and Talling

1965) is found in the eastern arm of the African (Eugster and Hardie

1978; Kaufman et al. 1990; Kavembe et al. 2014). The Lake basin is situated in the southernmost part of Kenyan Rift Valley (Pörtner et al. 2010). Lake Magadi has no river draining into it or draining out of it. This therefore makes the lake a closed system (Bützer et al. 1972). Lake Magadi is covered by a high precipitate of hydrated sodium carbonate salt (trona) which emanates rapid water evaporation (Kavembe et al. 2014). The lagoons of Lake Magadi are recharged by rare rainfalls and peripheral ephemeral geothermal springs. The well-known lagoons are the Fish Spring Lagoon, the South East Lagoons, the South West Lagoons and the Western Lagoons (Bützer et al. 1972; Goetz and Hillaire-

Marcel 1992). Some of the physiochemical conditions of the water in the lagoons around

sodium ion (340 ,(¹־Lake Magadi include: carbonate and bicarbonate ions (~ 200 mmol.1

,(¹־total carbon dioxide level (180 mM.L ,(¹־chlorine ion (100 mmol.1 ,(¹־mmol.1

and ,(¹־pH (~ 10.3), titration alkalinity (> 300mmol L ,(¹־conductivity (160 000 µmho.cm

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;Reite et al. 1974; Johansen et al. 1975; Eddy et al. 1981) (¹־osmolality (~ 563mOsm kg

Wood et al. 1989). The peripheral lagoons of Lake Magadi are located in the volcanically active Rift Valley so the temperature of the water can be as high as 46⁰ C (Coe 1966) and the oxygen content in the water fluctuates from hyperoxia (excessive content of oxygen) during daytime to hypoxia (oxygen deficiency) at night time (Narahara et al.

1996). The elevated oxygen content is caused by the abundance of (blue- green algae) in the water during photosynthetic activity (Maina 1999; Pörtner et al. 2010).

Lake Magadi and its peripheral lagoons are daily visited by many species of birds mainly flamingoes (Pörtner et al. 2010; Kavembe et al. 2014). Based on the environmental conditions, Lake Magadi is one of the most extreme aquatic environment on earth that supports fish life (Talling and Talling 1965).

In spite of Lake Magadi’s extremely harsh conditions, the Magadi tilapia (Alcolapia grahami Seegers and Tichy 1999 formerly Oreochromis grahami (Boulenger 1912) has developed some unique physiological adaptations for survival in this environment (Eddy et al. 1981). Alcolapia grahami is the only fish species which inhibits the isolated lagoons of greater Lake Magadi (Seegers and Tichy 1999). Among these rare traits, is the ability to extract oxygen air through their swim-bladder which is highly vascularized (Maina

1996). This strategy is achieved by the fish coming up to the surface of the water to gulp air via the mouth (Maina et al. 1996). Also, A. grahami completely eliminates nitrogenous metabolic end products in the form of urea from its body instead of (Randall et al. 1989). On the contrary, other freshwater teleost fishes excrete ammonia with minute quantity of urea (Randall et al. 1989).

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Large number of studies on the origin (Verheyen et al. 2003), the biology and ecology

(Fryer and Iles 1972), the morphology (Seegers and Tichy 1999), the physiology

(Narahara et al. 1996), and the genetics (Kavembe et al. 2014) of lacustrine cichlid fishes have been done especially those from the East African Lakes. However, less attention has been paid to the riverine Old World cichlids (Katongo et al. 2007).

1.6 Riverine Old World cichlids

Schwarzer (2011) suggested that the low attention on riverine cichlids could result from less diverse species with the similar morphology displayed by the riverine African genera. However, there are few species found in stretches of river which are good examples for speciation studies (Schwarzer 2011).

1.6.1 River Congo cichlid distribution

In Africa, the river with the most diverse species of fish is the (Teugels and

Guegan 1994). Approximately 700 known fish species are found in the Congo River, with most of them existing in the lower part of the river (Schwarzer et al. 2009). In particular, some genera such as , , , ,

Steatocranus, and are found in the lower part of the Congo River

(Schwarzer et al. 2009; Keck and Hulsey 2014). Besides, about 14 known species have

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been identified within the genus under the Haplotilapiine group, making it the most species-rich lineage in the Congo River (Schliewen and Stiassny 2006). From the Congo River, the spread of riverine cichlids (e.g. Haplochromines) began to spread into the Southern African water systems (Katongo et al. 2007).

1.6.2 Cichlid fishes of the southern African freshwater systems

Many studies have been done on the southern African riverine cichlids (Joyce et al. 2005;

Kocher 2005; Meyer 2005; Musilová et al. 2013). Four major lineages of African cichlids namely the riverine Haplochromine, the Serranochromine, the Sargochromine and the

Tilapiine are extensively distributed in the Southern African freshwater systems (Skelton

2001; Joyce et al. 2005; Musilová et al. 2013).

The Haplochromines are the most diversified lineage in southern Africa (Skelton 2001).

The cichlids of the Haplochromine lineage include seven genera including Astotilapia,

Chetia, Hemichromis, , , and

Thoracochromis (Kocher 2005). They are mostly found in the Kunene, the Limpopo the

Okavango and the River systems (Kocher 2005). From the ten species of the monotypic genera of Serranochromines found in Mainland Africa, seven species are widely spread throughout the Kunene, Kafue, Okavango and upper Zambezi river systems (Skelton 2001). All species in the Serranochromine lineage are exclusively

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predatory (Musilová et al. 2013). Also, Serranochromines use the strategy of mouth brooding for reproduction (Musilová et al. 2013).

Within the Tilapiines, there are two major genera namely Oreochromis and Tilapia found mainly in the Kunene, the Okavango and the Zambezi River systems (Skelton 2001). The

Sargochromines have seven species and they are exclusively endemic to southern

African freshwater systems (Skelton 2001). They mostly occur in the upper Zambezi along with the Okavango, the Kafue and the Kunene systems (Skelton 2001). In addition to knowledge on cichlid’s morphology, distribution, biogeography and habitat colonization, the need to further investigate their phylogeny is imperative. This will delineate cichlids pattern of speciation.

1.7 Phylogenetic relationships among cichlid assemblages

The phylogeny of cichlids is poorly investigated, especially regarding factors such as speciation and phenotypic divergence (Chakrabarty 2006; Genner et al. 2007; Hulsey et al. 2010; Sturmbauer et al. 2010; Gonzalez-Voyer and Kolm 2011; Hoerner 2011; López-

Fernández et al. 2013). Other fundamental factors such as identification combined with the vast evolutionary accomplishment of radiations when fully known could increase the knowledge on the speciation processes (Coyne and Orr 2004). Hence, it is imperative to reanalyse the phylogeny of cichlids to discover cryptic diversity (Coyne and Orr 2004).

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Based on morphological (Stiassny 1991) and molecular analyses (Sparks and Smith

2004; McMahan et al. 2013) the phylogeny of cichlids is monophyletic. Cichlids belong to the family cichlidae (McMahan et al. 2013) which is divided into four subfamilies, namely the Etroplinae, the , the and the (Sparks and Smith 2004; McMahan et al. 2013). The Etroplinae and the Ptychochrominae subfamilies make up two percent (with 31 known species) among the extant species of cichlids (Eschmeyer and Fong 2015). Based on their economic importance, within the subfamily Etropinae, two genera Etroplus and are most commonly known in south-Asia (India and Sri Lanka) and Madagascar inland waters (Sparks and Smith 2004;

Sparks 2008).

Among the cichlid sub families, New World cichlinae (542 identified species) and Old

World Pseudocrenilabrinae (1103 valid species) contain about 98% of the entire extant cichlids (Eschmeyer and Fong 2015), making these groups the most diversified groups in the family. There are three mega-diverse lineages (, Geophagini and

Heroini) as well as four basal smaller lineages (Astronotini, , Cichlini and

Retroculini) identified in New World cichlinae (Musilová 2011; McMahan et al. 2013).

Using mitochondrial (COI and 16S) and nuclear (histone H3 and Tmo4c4) genes,

McMahan et al. (2013) established nine lineages including Australotilapiini,

Boreochromini, Chromidotilapiini, Etia, Hemichromini, Heterochromini, Oreochromini,

Pelmatochronini and Tylochomini in African Pseudocrenilabrinae. The well-known East

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African radiation that is a leading model of adaptive radiation diversified from the

Australotilapiini group (Muschick et al. 2012). Also, vast numbers of Old World cichlids diversified mostly from the Oreochromini (75 species) and Australotilapiini (883 species) lineages (McMahan et al. 2013).

On the contrary, there have been challenges in resolving the phylogenetic relationships of African cichlids (Terai et al. 2003; Seehausen 2006). For example, controversy on monophyly of the African cichlids is still a debate because the position of Heterochromis has not been fully resolved based on a molecular approach (Keck and Hulsey 2014). This argument was based on whether Heterochromis is closely related to either monophyletic

African cichlids or the monophyletic clade of Neotropic cichlid species (Klett and Meyer

2002; Chakrabarty 2004).

The origin of the East African cichlid radiation is randomly positioned in the enormous lineage of Tilapiines using short interspersed elements (SINEs) found in the nuclear genome of these fishes (Terai et al. 2003). According to Klett and Meyer (2002);

Schliewen and Stiassny (2003) and Schwarzer et al. (2009), the Tilapiine group are commonly considered to be of paraphyletic origin. They comprise of a small number of species-rich and phenetically alike genera such as Oreochromis, and

Tilapia, with more than a few less species-rich and in rare instances monotypic genera such as Alcolapia, , , , , Steatocranus, and

Tristramella (Schliewen and Stiassny 2003). Although, the phylogenetic relationship

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within Haplotilapine lineage is yet to be fully resolved (Schwarzer 2011), the phylogeny of the Haplotilapine group comprising of the Boreotilapiines and Austrotilapiines has been investigated and revised based on nuclear and ND2 mitochondria DNA by Dunz and

Schliewen (2013).

Discovery of cryptic species, rate of evolutionary transformation, timing of rapid speciation events and phylogenetic divergence can be evaluated using molecular techniques such as DNA barcoding (Wong and Hanner 2008; Schwarzer 2011).

1.8 DNA barcoding – a molecular tool for species identification

Mayden (1997) and Pedial et al. (2010) inferred that radiation of species is achieved independently within lineages of populations or metapopulations. However, there are debates that cover the limit of speciation success (April et al. 2011). Over the years, taxonomists have used traditional techniques such as morphology and ecology to identify species (Robbins 1991; Pritchard and Mortimer 1999), but taxa with high morphological similarities are always difficult to distinguish. Molecular techniques have been employed to provide a more resolute power in species identification where morphological characters prove abortive (Radulovici et al. 2010). Furthermore, it is difficult to identify or classify organisms especially in scenarios where there is a phenotypic resemblance among unrelated species caused by convergent evolution (Lorenz et al. 2005). Biodiversity of

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most extant species are flawed by morphological characteristics alone (Lorenz et al.

2005).

Previously, taxonomist, have tried to establish an orderly pattern of fish diversity based on characteristics of the internal and external body structures (Kocher and Stepien 1997).

Following this conventional method, taxonomist achieved remarkable success in the identification of evolutionarily related lineages among groups. Despite the achievements of the conventional approach, species groups with limited morphological differences have been difficult to characterise (Kocher and Stepien 1997). As such, molecular techniques have revolutionised this field in several ways (Kocher and Stepien 1997). For instance, molecular approaches have supported the conventional method in the placement of taxonomic units, with a number of cases showing contrasting patterns between them

(Kocher and Stepien 1997). Further, molecular techniques have been useful in the discovery of cryptic diversity (Avise 1994).

The use of molecular genetic markers started in the mid-ninety’s and since then, there have been advancement of techniques in studying organisms’ similarities at molecular level (Park and Moran 1994). The application of this technique on fish began with the discovery of alloyzme (functionally parallel but distinguishable forms of enzymes that are the products of different alleles at the same locus) polymorphism in fish systematics which entails comparison of similarities and differences in net electric charge of protein polymorphism (Kocher and Stepien 1997). The studies of allozyme and isozyme have

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effectively been used by fish geneticist to assess questions on population genetics and stock divergence in fishes (Chaturvedi et al. 2011; Zhigileva et al. 2013). On the contrary, one of the main disadvantages of allozyme techniques is that non-homologous alleles of similar electric charge travel along the same point in the gel, creating difficulty in bands interpretation (Kocher and Stepien 1997).

A more advanced technique in molecular studies is using nuclear and mitochondrial

DNAs to address genetic questions. These methods have shown great properties in the reconstruction of phylogenetic relationships (Avise 1994). Selected nuclear DNA such as AFLP (amplified fragment length polymorphism) have been applied to study the evolutionary processes of the cichlid genus Tropheus in Lake Tanganyika (Egger et al.

2007). Nuclear rDNA (i.e. DNA sequence that codes for ribosomal RNA) spacers have been used in the study of interspecific and subspecific levels (lower taxonomic levels) among some fish group (Phillips and Oakley 1997). ITS-1 (nuclear internal transcribed spacers) found in the ribosomal region have been used to address deeper divergences within blennioid fishes (Stepien et al. 1993).

One advantage of mitochondrial genome is that it possess a clonal inheritance e.g. the mitochondrial genes of fish are nonrecombining and haploid (Kocher and Stepien 1997).

Another advantage of using mitochondrial DNA is that in comparison to other nuclear genes, mtDNA evolves faster and permits the identification of informative phylogenetic characters among closely related groups (Kocher and Stepien 1997).

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Creating fast and correct molecular discoveries by applying reliable novel tools might be capable of resolving biased interpretations of biodiversity. The innovation of molecular techniques has unlocked great possibilities in the knowledge of biodiversity (Packer et al.

2009). Among other molecular methods, a novel mitochondrial-technique called “DNA barcoding” was proposed for better resolution of biological identification, whether at species level, within phyla or from the whole kingdom (Hebert et al. 2003). The proposal of DNA barcoding, which targets mitochondrial cytochrome c oxidase subunit I

(COI) gene, has got its weaknesses and strengths just as other molecular techniques have (Radulovici et al. 2010). A weakness of COI was presented in the results of Song et al. (2008), where DNA barcoding was ambiguous in resolving mitochondrial pseudogenes in some insects. It was also recorded by Siddall et al. (2009) that DNA barcoding was unable to achieve the goal of their research due to amplified contaminants with universal primers. Also, it is difficult for DNA barcoding to identify many organisms where it involves functional groups (Radulovici et al. 2010) and conditions of introgression caused by mitochondrial divergence (Kemppainen et al. 2009).

Irrespective of DNA barcoding’s limitations, recent studies still suggest that this novel tool is a potent tool for investigating animal biodiversity (Wong and Hanner 2008; Ward et al.

2009), species identification in all life stages, discrimination between sexes and potential tool for cryptic species (indistinguishable biological groups based on morphological criteria) identification (Radulovici et al. 2010).

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In addition, for better resolution of the gap between intraspecific and interspecific variations (Lin et al. 2009), the conserved sequences of COI within the mitochondrial gene allows ‘universal’ primers to be used across multiple divergent taxa. Cytochrome c oxidase subunit I gene also has high degree of phylogenetic signal compared to other mitochondrial genes such as 12S or 16S ribosomal DNA (Lorenz et al. 2005). In the field of forensics, DNA barcoding has been proven successful in the investigation of market substitution and authenticity testing of food product (Wong and Hanner 2008), unlawful poaching (Eaton et al. 2009) and species separation (Wilson-Wilde et al. 2010).

The use of DNA barcoding has demonstrated its usefulness in accelerating species recognition, phylogeographic studies, biodiversity estimations and conservation efforts

(Radulovici et al. 2010; Nwani et al. 2011). According to Ward et al. (2009), the initiative of fish barcode of life universally presented DNA barcoding as a fast, precise, economical and widely appropriate technique for identification of species. In particular, DNA barcoding was used to solve the authentication of Oreochromis mosambicus, which was being sold as white tuna sushi (Thunnus alalung) (Wong and Hanner 2008). There have been many studies on freshwater fish species based on species identification and COI divergence with the aid of DNA barcoding (Hubert et al. 2008, Valdez-Moreno et al. 2009;

Van der Bank et al. 2012).

Recently, efficiency of DNA barcoding has been shown in many studies of freshwater fish

(Nwani et al. 2011). In addition, with the aid of COI gene together with other mitochondrial

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and nuclear genes, the phylogeny and biogeography as well as pattern of temporal diversification across the biodiversity of cichlids have been delineated (Sparks and Smith

2004; McMahan et al. 2013).

The insight to structuring this project was obtained from the revised classification and molecular phylogenetic study of the Haplotilapiine lineage by Dunz and Schliwen (2013).

Three genomic groups from Lake Magadi (Little Magadi, Fish Spring Lagoon and the rest of Lake Magadi) were identified within the population of Alcolapia grahami using microsatellite and mitochondria DNAs (ND2 gene and control region) markers (Kavembe et al. 2014). To date, there has not been any molecular study done on Thoracochromis albolabris using COI to establish its phylogenetic relationship within the family Cichlidae.

1.9 Hypothesis and objectives of the study

Hypothesis:

 Will COI give a clear identification of some cichlids from the Old and New world?

 Can COI gene identify evolutionary pattern in Old World (African) versus New

World (Neotropical) cichlids?

Objectives:

The goal of this study was to determine the potential of DNA barcoding in providing insight into the identification of some cichlid fishes. The following are the specific objectives: Page | 23

 to re-assess the efficacy of DNA barcoding (COI gene) for species identification of

family Cichlidae

 assessing the presence or absence of barcode gap

 using COI gene to identify the evolutionary pattern in Old World (African) versus

New World (Neotropical) cichlids

 to investigate whether different isolated populations of Alcolapia grahami have

speciated

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2. MATERIALS AND METHODS

2.1 Locations and habitats of species sampled

For the location and habitat of the Magadi tilapia Alcolapia grahami refer to section 1.4.4 in this study. The specimens of A. grahami used in this study were caught from Fish

Springs Lagoon (Latitude -1.892⁰ and longitude 36.303⁰ ) and South west lagoon

(Latitude -2.001⁰ and longitude 36.232⁰ ) with the use of a seine net.

Thoracochromis albolabris and T. buysi are endemic to the Kunene systems. For the purpose of this study, which is to identify T. albolabris and T. buysi at molecular level using DNA barcoding and then include the generated DNA sequences in BOLD systems database for the first time. Specimens of T. albolabris and T. buysi were caught by the use of hook and earthworm from the Kunene River system situated on the border between Angola and Namibia. The sampled site of T. albolabris was observed to have crevices and rocky environment. This site is more distant from the South Atlantic Ocean with slow water current as compared to sample site for T. buysi. On the contrary, site of T. buysi was observed to be surrounded with sandy environment with few vegetation at the river bank. It is much closer to the South Atlantic Ocean and water current is fast flowing during the rainy season.

2.2 Sampling of taxa

To assess the potential of the mitochondrial gene, cytochrome c oxidase subunit I (COI) gene on Cichlidae for this study, sequences of cichlids including some representatives of subfamilies Etroplinae, African Pseudocrenilabrinae (Old World cichlids; Fig. 2) and

Ptychochrominae were generated and some were mined from BOLD. COI sequences from two populations of Alcolapia grahami (from two peripheral lagoons around Lake

Magadi, Kenya, namely Fish Spring and South West Lagoons) were generated for the

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first time (Fig. 3). The accession numbers are deposited on Barcode Of Life Database

(BOLD; www.boldsystems.org). Also for the first time, COI sequences of two species from

River Kunene, Namibia, Thoracochromis albolabris and T. buysi (Fig. 4) were also generated and accession numbers were deposited on BOLD. Furthermore, sequences of

New World cichlids including some representatives of the subfamily Cichlinae were also mined from BOLD. DNA extraction, polymerase chain reactions (PCR), and sequencing of COI region were done at the African Centre for DNA Barcoding (ACDB) at the

University of Johannesburg, South Africa.

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Fig . 2. Map of Africa indicating the distribution of some African cichlids. Each coloured circle represents the sites where cichlids were collected. The sizes of circle does not correspond to number of cichlids, however, they are of different sizes to accommodate other genera sampled at same point. Some genera such as Serranochromis and Oreochromis have Pan-African distribution (i.e. they occupy different water systems across southern Africa countries). On the contrary Alcolapia is endemic to Kenya.

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Fig. 3. Distribution of Magadi tilapia (Alcolapia grahami). Map of Kenya showing the location of Lake Magadi (brine water / trona) basin indicating the locations of two peripheral lagoons sites (Fish Springs Lagoon and South West Lagoon indicated) from which the fish were collected (adapted from Maina et al. 1996).

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2

 N 50 km

2 1

N 200 km

1  N 50 km

Fig. 4. Distribution of genus Thoracochromis in southern Africa (a) map of Angola and Namibia showing the locations and sites of two endemic species of T. albolabris (blue square with number one; -17.417⁰ , 14.019⁰ ) and T. buysi (blue square with number two; -17.272⁰ , 11.786⁰ ) found in Kunene River along both countries borders, from which the fish were collected. (b) This is a Kunene system located around Namibia and Angola borders and it is supported by rocky environment and vegetation. It is also far away from the South Atlantic

Ocean (c) this is around the Kunene mouth of Namibia and Angola borders which flows into the South Atlantic Ocean and it is supported by sandy environment and few vegetation. Maps were generated by Google scribble map (https://www.scribblemaps.com ) and modified manually.

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2.3 DNA extraction

The DNA extraction process involves isolation of nuclear and/or organelle DNA from other biological (carbohydrate, lipid and protein) materials contained in the cell (Dahm 2008).

In the late 20th century, different methods such as grinding, precipitation and washing were employed to isolate DNA (e.g. Maniatis et al. 1982). These methods may include toxic materials (such as chloroform) and laborious steps. However, in this modern time, there have been invention of many commercial kits with protocols which delivers safer and faster DNA extraction (see Nishiguchi et al. 2002). The following manufacturers’ recommended protocol developed from NucleoSpin® Tissue kit (Macherey-Nagel) was used in this study.

Prior to DNA extraction, fin tissues were collected from 10 individuals of A. grahami and six individuals of T. albolabris preserved in absolute ethanol in a properly labelled microplate. The microplate was kept at -20⁰C until it was processed. Water bath was switched on and set at 56⁰C. The fish tissues were weighed and placed in different labelled 1.5 mL Eppendorf® Safe-Lock microcentrifuge tubes. The weight of the tissues varied from 0.002 - 0.078g. During DNA extraction process, each microcentrifuge containing fin tissue, 180 µL of buffer T1 and 25 µL of proteinase K solution were added to separate DNA from all materials inside the cell, vortexed for 15 seconds to mix the solutions and tissue samples. Proteinase K is an enzyme capable of digesting proteins.

The samples were then incubated in a water bath at 56⁰C for about 9 hours. This process breaks up fish tissue. The samples were removed from the water bath and vortexed for

15 seconds. Next, 200 µL buffer B3, was added to the samples which were later vortexed

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vigorously for 35 seconds. These lysed samples were further incubated at 70⁰C for 10 minutes and vortexed for 15 seconds. This process further breaks proteins into DNA molecules. Then, 210 µL of absolute ethanol (100%) was added to samples for DNA precipitation. Samples were carefully transferred with the use of a pipette into prepared labelled NucleoSpin® tissue columns (the column contain silica membrane which acts as semipermeable membrane, i.e. it allows micromolecules such as H₂O to diffuse through while retaining macromolecules such as DNA) held in collection tubes. During this process pipette tips were changed as each sample was being transferred. This is to avoid

DNA contamination. Afterwards samples are placed in a centrifuge and spun at 11 000 x g for one minute to bind DNA to silica membrane. The collection tubes containing flow- through (solution collected in the collection tubes) were discarded. The columns were placed in new collection tubes. These columns were washed twice with buffer BW and buffer B5. In the course of the first wash, 500 µL of buffer BW was added in the columns and centrifuged at 11 000 x g for one minute. The flow-through in the collection tube was discarded and the columns were placed back into the same collection tube. In the second wash, 600 µL of buffer B5 was added in each columns and also centrifuged at 11 000 x g for one minute. The flow-through in the collection tube was thrown away and the columns were placed back into the same collection tube. To remove residual ethanol with other solutions, the columns were centrifuged at 11 000 x g for one minute and the collection tubes were finally discarded. In order to get clean DNA retained from the silica membrane, NucleoSpin® tissue columns were placed into properly labelled 1.5 mL

Eppendorf® Safe-Lock microcentrifuge tubes. 100 µL of buffer BE was added into each column and was left to incubate under room temperature for one minute, then centrifuged

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at 11 000 x g for one minute. Finally, clean DNA products were collected in the 1.5 mL microcentrifuge tubes and the columns were discarded. DNA products were quantified with the aid of a spectrophotometer and viewed after running gel electrophoresis.

Gel electrophoresis is the process where DNA is driven from one polar region to the other polar region with the aid of electric current. DNA migrated towards the positively charged pole when electric current was initiated. This implies that DNA is negatively charged. To monitor the movement of DNA on the gel, loading gel dye was used to stain DNA before loading into the gel. Two types of gels can be used for electrophoresis (namely agarose gel and polyacrylamide gel) depending on DNA length. For shorter DNA length polyacrylamide gel can be used and agarose gel is used for longer DNA length. For this study, 1.5% agarose gel was used, the stained DNA was visualised using ultra-violet light after electrophoresis. The visible products were selected for DNA amplification using a

PCR machine.

2.4 PCR amplification

DNA amplification of the COI sequences (approximately 650-bp long) were achieved by the PCR using these sets of primers:

(a) Co1-Fish.F1 5'-TCAACCAACCACAAAGACATTGGCAC-3' and

(b) Co1Fish.R1 3'-TAGACTTCTGGGTGGCCAAAGAATCA-5'.

The PCR reactions were done in a total volume of 25 µL, however two different master mixes were prepared with different protocols. The first master mix consisted of 12.5 µL of top taq, 0.8 µL of BSA, 0.3 µL of each primer and 10.6 µL of dH₂O. The second master

Page | 32

mix contains 22.5 µL of red pre-mix, 0.8 µL of BSA, 0.3 µL of each primer and 0.1 µL of dH₂O. DNA templates ranged from 1-3 µL depending on the quality of DNA product quantified from spectrophotometer and viewed on an agarose gel. The following thermocycling conditions were used to amplify COI region of the mitochondrion gene beginning with pre-melting for two minutes at 95⁰C, denaturation at 94⁰C for 30 seconds, annealing at 52⁰C for 30 seconds, extension at 72⁰C for one minute followed by a final extension at 72⁰C for 10 minutes for 35 cycles and a hold at 4⁰C (Steinke and Hanner

2011). PCR products were visualised on a 1.5% agarose gel using ultra violet light. Visible products were cleaned.

2.5 PCR reaction cleanup

According to Simon et al. (1991), PCR cleanup was implemented to eliminate extra nucleotides, primers and salt prior to sequencing. 100 µL of buffer PB was added to the

PCR reaction. Silica columns were prepared and placed into a vacuum manifold. The samples were loaded into the silica columns and vacuum was applied. Afterward, the columns were washed by addition of 750 µL of buffer PE and applied vacuum again. The columns were transferred to a 200 µL collection tube and centrifuged at 11 000 x g for one minute. Next, silica columns were placed into a clean 1.5 mL Eppendorf® Safe-Lock microcentrifuge tubes. Finally, to elute the DNA, 20 µL of elution buffer was added directly onto the silica membranes. Then left for 20 minutes at room temperature to incubate.

Centrifuge was applied at 11 000 x g for two minutes. The cleaned PCR products were quantified with the aid of a spectrophotometer and viewed after running gel electrophoresis. The visible products were selected for cycle sequencing.

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2.6 DNA cycle sequencing

Cycle sequencing reactions were done in a total volume of 10 µL. Both forward and reverse reactions were carried out separately, with each sample containing 0.3 µL of big dye terminator, 1.5 µL of 5x sequencing buffer, 0.3 µL of same PCR primer and 1 µL of cleaned PCR products. Thermocycling condition was the same as PCR. The cycle sequencing reactions were cleaned using EtOH-NaCl (ethanol-sodium chloride) and the products were detected using an ABI 3130x1 genetic analyzer. Cycle sequencing was achieved following ACDB protocol for cycle sequencing.

2.7 DNA sequence analysis

ABI files of both forward and reverse sequences for each sample generated were assembled, trimmed and edited using Sequencer vs 4.6 (Genecodes Corp, Ann Arbor,

Michigan, USA). The sequence assembly for each sample was generated using the same

Sequencer. All sequences including the sequences generated and downloaded were aligned using Multiple Sequences Comparison by Log-Expectation (MUSCLE vs 3.8.3.1;

Edgar 2004) in Seaview vs 1.0.10.0. Additionally, a final quality control measure was done, where aligned sequences were manually aligned.

2.8 DNA Barcoding

Half a decade ago BRONX (Barcode Recognition Obtained with Nucleotide eXposés) identification tool was invented (Little 2011). This novel tool describes observable differences found between intraspecific taxon and groups interspecific taxa in a hierarchical order. BRONX helps to lower the chance of misidentification which may have

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originated from short sequences, shared haplotypes and imperfect identification from downloaded sequences. In other words BRONX is important in the improvement of this dataset. This tool was applied to the entire dataset including generated and mined sequences, in order to filter-out the problematic sequences and distinctly identify each taxon before further barcoding analyses where done.

The performance of COI gene in species delimitation was tested at species level under both genus and family (entire dataset) levels. The approach of delineating the barcoding potential was achieved following two criteria including: (a) the occurrence of barcoding gap (Meyer and Paulay 2005), and (b) discriminatory power.

The evaluation of barcode gap was attained by two methods. Firstly, the use of the mean, median and range of intraspecific genetic distance (genetic differences within species) against interspecific genetic distance (genetic variation between species) (Meyer and

Paulay 2005). Secondly, the approach of Meier et al. (2006) in delineating barcode gap which was achieved by matching the lowest interspecific distance against the highest intraspecific distance. In addition, both genetic distances were analysed using a model called the Kimura 2-parameter (K2P) (Posada and Buckley 2004). This test was achieved by SPIDER (SPecies IDentity and Evolution in R) in R package vs 1.1-1 (Brown et al.

2012).

Two methods such as Near Neighbour and Best Close Match were inducted for the computation of the discriminatory power of COI gene (Meier et al. 2006). To achieve a

Page | 35

high-quality barcode result, there should be a very high rate of identification of correct species giving to the highest fraction of sequenced DNAs which in turn must correspond to the name of species (Meyer and Paulay 2005). Prior to these distance-based analyses, labelling of sequences was done corresponding to species name. Also, prior to carrying out these analyses, optimised thresholds were generated using the “localMinima” function executed in Spider in R package vs 1.1-1 (Brown et al. 2012).

2.9 Phylogenetic tree reconstruction

Reconstruction of phylogenetically delimitated clades in the phylogeny from all taxa sequences were used to test the DNA barcoded species. Distinctive haplotypes of all sequences were first generated using DnaSP vs 5.10.01 (Librado and Rozas 2009) for reconstruction of phylogenetic trees. Based on the phylogenetic tree of Acanthomorpha

(fishes with spiny rays) by Wainwright et al. (2012), Embiotoca jacksoni (Embiotocidae) and Abudefduf saxalitis (Pomacentridae) were used as out-groups. These families were also reported to be more closely related to Cichlidae based on mitochondrial gene (Turner

2007). Maximum-parsimony (MP) tree was reconstructed using PAUP* version 4.0 b2a

(Swofford 2003). MP trees were generated using heuristic search under the following conditions: swap on best trees; 1 000 random sequence, holding 10 trees per replicate; branch swapping using tree-bisection-reconnection (TBR) function, saving only 10 trees for every 1 000 replicates and swaping only on best trees. MP tree was recovered using tree drawing option and tree was printed after rooting. One method adopted for rooting unrooted tree was to make the in-group monophyletic and the out-group a monophyletic sister group to the in-group. Nodes for support were performed by the application of

Page | 36

bootstrap (BP) values: when BP value is greater than 70%, it signifies strong support

(Wilcox et al. 2002). For a more robust result, Bayesian approach as implemented in

MrBayes 3.1.2 was used to reconstruct a phylogenetic tree of the family Cichlidae

(Ronquist and Huelsenbeck 2003). Prior to the Bayesian tree reconstruction, the best-fit model of DNA sequence evolution was done using Modeltest 3.7.mac9 (Posada 2008) following the Akaike information criterion (Posada and Buckley 2004). The best-fit model generated amongst all 56 model tested was TrN + I + G (Tamura-Nei; proportion of invariable sites; gamma distribution) which is used to generate the Bayesian phylogenetic tree (Posada and Crandall 2001).

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3. RESULTS

3.1 Extraction of DNA and amplification of COI gene

The kit used to extract DNA from fin tissues was effective and successful. It produced

good clear smear of DNA products as seen on the 1.5% agarose gel (Fig. 5). Further, the

amplification of DNA strands to generate copies of mitochondrial DNA, particularly COI

sequences was achieved following the PCR and clean PCR protocols mentioned in this

study (Fig. 6).

A C DB E GF H I J K L M NO P

-

+

Fig. 5. Gel electrophoresis products of clean DNA of two African cichlid fishes viewed in 1.5% agarose gel with the aid of ultra-violet light. A to E are cleaned DNA products of Alcolapia grahami from Fish Spring Lagoon (Lake Magadi, Kenya), F to J are cleaned DNA products of A. grahami from South West Lagoon (Lake Magadi, Kenya), K to P are cleaned DNA products of

Thoracochromis albolabris from Kunene River, Namibia. Little arrows are pointing at the spots DNA were loaded into agarose gel. Bigger arrow shows the migration of DNA from negative (-) pole towards positive (+) pole.

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A B C D E F G H I J K L M N O P i -

+

ii A B C D E F G H I J K L M N O P

-

+

Fig. 6. Gel electrophoresis products of i) PCR products ii) Clean PCR Products of two cichlid

species visualised in 1.5% agarose gel with the aid of ultra-violet light. A to E are products of Alcolapia grahami from Fish Spring Lagoon (Lake Magadi, Kenya), F to J are PCR

products of A. grahami from South West Lagoon (Lake Magadi, Kenya), K to P PCR

products of Thoracochromis albolabris from Kunene River, Namibia. Little arrows point at the wells PCR were loaded into agarose gel. Bigger arrow shows the migration of DNA from

negative (-) pole towards positive (+) pole. PCR and clean PCR were ran at different times

on different 1.5% agarose gels.

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3.2 Sequence mining and filtering

The entire dataset for the barcoding analyses include, 159 generated (22 newly generated) sequences and 121 retrieved sequences from BOLDsystems. These sequences were aligned using MUSCLE (Edgar 2004). Next, BRONX (Barcode

Recognition Obtained with Nucleotide eXposés; Little 2011) was used to evaluate the sequences for proper identification thus reducing and/or eradicating incorrectly identified sequences which might have arisen from shared haplotypes by integrating observed intraspecies variability into the scoring system. Out of the 280 sequences queried by

BRONX, 18 sequences returned as ambiguous sequences not corresponding uniquely to the named taxon (i.e. having two or more matches); which were removed from the analysis. On the other hand, 262 sequences were observed as unique and correct sequences with match to their named taxa.

3.3 DNA barcoding

The dataset barcoded following BRONX includes 262 specimens of cichlid fishes. These specimens represented 52 species, distributed across 16 genera. The final lengths of COI sequences after alignment was 652 base pairs (bp) comprising 23.4% A, 29.7% C, 17.9%

G and 29% T. These variations in base pairs could have led to morphological variations among species, hence leading to identification of species (Ball et al. 2005).

Optimised threshold of genetic distance for species delimitation was generated for individual genus and at family level (Table 1). These threshold values were used to test discriminate power of COI sequences in best close match method. Apart from Alcolapia

Page | 40

(threshold = 0.11%), Pseudocrenilabrus (threshold = 0.16%), (threshold =

0%) Serranochromis (threshold = 0.77%) Thoracochromis (threshold = 0.07%) and

Tristramella (threshold = 0.09%) which exhibited threshold lower than 1%, the rest of the genera showed threshold values greater than 1% (Table 1). The genus Etroplus had the highest threshold value of 9.44%, followed by Hemichromis (threshold = 8.83%) and

Cichla (threshold = 8.24%). At the family level, a threshold of 2.86% was generated (Table

1).

From the pairwise genetic distance analyses that were performed among the 16 genera, the ranges and mean interspecific distances were both greater than those of intraspecific distances (Table 1). Three genera including Alcolapia (i.e. 10 individuals),

Pseudocrenilabrus (13 individuals) and Tristramella (7 individuals) did not have any range nor mean interspecific distance. In addition, to confirm the test that showed no range and mean interspecific variation within the A. grahami group. One population of Alcolapia grahami was given a fictional name whereas the other population was left with its original name and ran the pairwise genetic distance analyses again. Still there was no variation

(result not shown). This observation might have been as a result of single species and fewer individuals included in these genera. For instance, each of the three genera was represented by a single species, which implies that a lot of within species variation was not captured, hence the lower levels of genetic distances among these genera. However, there is variation within the above mentioned genera due to the generated values of their intraspecific distances (Table 1). The genera that exhibited elevated ranges in interspecific genetic distances were Cichla (8 individuals; 5 species), (25

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individuals; 2 species), Etroplus (5 individuals; 2 species), Hemichromis (16 individuals;

4 species), Herichthys (11 taxa; 4 species), Oreochromis (43 individuals; 5 species),

Sargochromis (6 individuals; 3 species), Sarotherodon (13 individuals; 3 species),

Serranochromis (21 individuals; 5 species), Steatocranus (7 individuals; 3 species),

Thoracochromis (13 individuals; 2 species), Tilapia (63 individuals; 6 species) and

Tylochromis (19 individuals; 6 species). These genera also showed much variation within species except for Cichla which exhibited no intraspecific variation within its five species.

Higher gap values were seen in genera with more numbers of individuals and species

(e.g. Oreochromis) compared to genera (e.g. Tristramella) with only one species and few individuals. Assessing the efficacy of DNA barcoding on COI sequences, different thresholds were established on different groups (Table 1).

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Table 1: Barcoding summary on the genera with at least four sequences. K is Tajima’s index of sequence divergence

Genus Number of Number K Range (inter) Mean inter Range (intra) Mean intra Threshold (%) sequences of (±SD) (±SD) species Alcolapia 10 1 0.0022 0 0 0 - 0.0050 0.0027 ± 0.0015 0.11

Cichla 8 5 0.0679 0.0179 - 0.1506 0.0978 ± 0.0534 0 0 8.24

Coptodon 25 2 0.0096 0.0217 - 0.0267 0.0232 ± 0.0015 0 - 0.0049 0.0012 ± 0.0013 1.24

Etroplus 5 2 0.0945 0.1971 - 0.2141 0.2060 ± 0.0063 0 - 0.0127 0.0063 ± 0.0058 9.44

Hemichromis 16 4 0.1009 0.0031 - 0.2196 0.1513 ± 0.0754 0 - 0.2238 0.0322 ± 0.0699 8.83

Herichthys 11 4 0.0352 0.0015 - 0.0744 0.0422 ± 0.0246 0 - 0.0666 0.0239 ± 0.0268 5.27

Oreochromis 38 5 0.0332 0 - 0.0642 0.0439 ± 0.0150 0 - 0.0596 0.0072 ± 0.0131 2.51

Pseudocrenilabrus 13 1 0.0017 0 0 0 - 0.0047 0.0017 ± 0.0016 0.16

Sargochromis 6 3 0.0039 0.0015 - 0.0078 0.0050 ± 0.0018 0 - 0.0046 0.0023 ± 0.0016 0

Sarotherodon 13 3 0.0286 0.0086 - 0.0613 0.0489 ± 0.0150 0 - 0.0116 0.0041 ± 0.0047 2.98

Serranochromis 10 3 0.0061 0 - 0.0173 0.0070 ± 0.0065 0 - 0.0141 0.0025 ± 0.0044 0.77

Steatocranus 5 3 0.0364 0 - 0.0664 0.0449 ± 0.0182 0 - 0.0664 0.0157 ± 0.0283 2.18

Thoracochromis 13 2 0.0015 0.0015 - 0.0052 0.0029 ± 0.0010 0 - 0.0017 0.0006 ± 0.0008 0.07

Tilapia 63 6 0.0560 0.0062 - 0.1643 0.0895 ± 0.0650 0 - 0.0118 0.0006 ± 0.0017 6.37

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Genus Number of Number K Range (inter) Mean inter Range (intra) Mean intra Threshold (%) sequences of (±SD) (±SD) species Tristramella 7 1 0.0004 0 0 0 - 0.0016 0.0005 ± 0.0008 0.09

Tylochromis 19 6 0.0572 0.0062 - 0.1199 0.0799 ± 0.0386 0 - 0.0077 0.0018 ± 0.0026 6.63

Family 262 52 0.1069 0 - 0.2772 0.1445 ± 0.0533 0 - 0.2238 0.0030 ± 0.0130 2.86

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Other than evaluating these genetic variations, Tajima’s (1983) index (K) of sequence divergence was also calculated for each genus, which establishes evolutionary relationship among COI sequences sampled. It was observed that Hemichromis exhibited the highest Tajima’s index of divergence (K = 0.1009) whereas Tristramella showed the lowest divergence index (K = 0.0004). The K of the entire dataset was calculated to

0.1069767, which further established a range of interspecific distance from 0 to 0.2772 with a mean of 0.1445. This interspecific distance at familial level is greater than its intraspecific distance which ranged from 0 to 0.2238, with a mean distribution of 0.0030

(Table 1).

The interspecific versus intraspecific distances for some genera and at family level exhibited a clear overlap between both distances (Fig. 7). This implies the existence of barcode gap in the dataset. When the smallest interspecific distance against the largest intraspecific distances were compared after using the Meier et al. (2008) approach, the result further supported the existence of barcode gap in the dataset. The proportion of sequences with barcode gap varied at each genus test. Notably, some genera such as

Alcolapia, Cichla, Coptodon, Etroplus, Pseudocrenilabrus, Sarotherodon, Tristramella and Tylochromis showed no proportion of sequences with gap (Table 2). However, the genera that exhibited proportion of sequences with gap include Oreochromis (86%) with highest gap, followed by Serranochromis (76%) and Herichthys (64%). The lowest proportion of sequences with gap was found in Sargochromis (17%) (Table 2). The proportion of sequences with gap observed at the family level was 25% (Table 2).

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a b

Fig. 7. Barcode gap evaluation of cichlid family represented in this dataset. (a) Boxplot of the interspecific and intraspecific genetic distances, demonstrating the existence of a barcode gap. The protruded lines from the box shows the first (bottom) and third (top) quartiles, the median is represented by the horizontal line and the little circles are outliers. (b) Barplot of the barcode gap for the 280 cichlid individuals. The black lines indicate where the lowest interspecific distance (top of line value) is greater than the highest intraspecific distance (bottom of line value), hence showing presence of barcode gap; the red lines show where this pattern is reversed.

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Table 2: Percentage barcode gap in entire dataset applying the Meier et al. (2006) method.

Genus Number of sequences Proportion of sequences with without gap gap (%) Alcolapia 10 0 Cichla 8 0 Coptodon 25 0 Etroplus 5 0 Hemichromis 6 63 Herichthys 4 64 Oreochromis 6 86 Pseudocrenilabrus 13 0 Sargochromis 5 17 Sarotherodon 13 0 Serranochromis 5 76 Steatocranus 3 57 Thoracochromis 7 46 Tilapia 43 32 Tristramella 7 0 Tylochromis 19 0 Family 197 25

On the overall results, the performance of COI sequences on species identification varied with the methods applied. Across the three methods namely, near neighbour, best close match and BOLD identification criteria; individual genera such as Alcolapia, Coptodon,

Etroplus, Pseudocrenilabrus, and Tristramella all exhibited 100% correct identification

(Table 3). The genera Cichla and Hemichromis both showed 75% correct identification while Steatocranus had 43% identification across the three methods. However, there was more similarity between the near neighbour and best close match methods as Herichthys

(82%), Sargochromis (50%), Sarotherodon (92%) and Tylochromis (95%) individuals were correctly identified (Table 3). The genus Oreochromis (near neighbour = 95%; best close match = 86%; BOLD = 67%) showed variation of correctly identified specimen

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across the three methods. The genus Serranochromis also exhibited variation in COI sequences that were correctly identified by the near neighbour, best close match and

BOLD identification criteria having 43%, 29% and 5% respectively. Furthermore, the genus Tilapia had variations (near neighbour = 100%; best close match = 98%; BOLD identification criteria = 27%) in COI sequences that were correctly identified across the three methods used. Bold identification of 1% threshold provided the lowest rate of correct identification among the three methods applied at individual level. For instance,

Herichthys (64%), Oreochromis (67%), Sargochromis (0%), Sarotherodon (69%),

Serranochromis (5%), Tilapia (27%) and Tylochromis (79%) gave lower identification of correct individuals when compared against both near neighbour and best close match

(Table 3).

At the family level the near neighbour method gave discriminatory power of 93% COI sequences were identified as “correct”. Best close match also gave 93% correctly identified sequences of COI. BOLD identification criteria exhibited a much lower discriminatory power (60%) of COI sequences correctly identified. However, near neighbour method indicated 7% of the COI sequences as “False” i.e. incorrectly identified.

The best close match method indicated 1% proportion of the COI sequence was ambiguous (i.e. both correct and incorrect species are within the given threshold), 4% were incorrectly identified and 2% of the sequence was without identification (Table 3).

BOLD identification method exhibited 35% proportion of ambiguity, 3% incorrectly identified and 2% had no identification.

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Table 3. Identification efficacy of DNA barcodes applying distance based methods in percentage.

NEAR BEST CLOSE MATCH BOLD ID CRITERIA NEIGHBOUR Genus No No True False Ambiguous Correct Incorrect Ambiguous Correct Incorrect ID ID Alcolapia 100 0 0 100 0 0 0 100 0 0 Cichla 75 25 0 75 0 25 0 75 0 25 Coptodon 100 0 0 100 0 0 0 100 0 0 Etroplus 100 0 0 100 0 0 0 100 0 0 Hemichromis 75 25 6 75 6 13 6 75 6 13 Herichthys 82 18 0 82 18 0 18 64 18 0 Oreochromis 95 5 9 86 5 0 28 67 5 0 Pseudocrenilabrus 100 0 0 100 0 0 0 100 0 0 Sargochromis 50 50 0 50 50 0 67 0 33 0 Sarotherodon 92 8 0 92 8 0 23 69 8 0 Serranochromis 43 57 57 29 14 0 95 5 0 0 Steatocranus 43 57 14 43 29 14 29 43 14 14 Thoracochromis 100 0 0 100 0 0 100 0 0 0 Tilapia 100 0 0 98 0 2 71 27 0 2 Tristramella 100 0 0 100 0 0 0 100 0 0 Tylochromis 95 5 0 95 5 0 16 79 5 0 Family 93 7 1 93 4 2 35 60 3 2

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3.4 Outline of nucleotide variation

A total of 652 characters (bp) were considered in this analysis and 30 characters excluded

(missing bp). Of the remaining 622 included characters: all characters are of the type

“unord” and are equally weighted. Three hundred and sixty six characters remained constant throughout this analysis. Variable characters that were considered as parsimoniously uninformative amounted to 24 whereas 262 variable characters included were considered as parsimony-informative character in this analysis. Furthermore, gaps were treated as missing and optimization of character-state was treated with the command prompt DELTRAN (i.e. delayed transformation) in the phylogenetic analysis.

Out of 76 included taxa, two phylogenetic trees were generated from this maximum parsimony (MP) analysis. The trees were rooted using the out-group method. The out- groups included in this study were Abudefduf saxatilis (Linnaeus, 1758) and Embiotoca jacksoni Agassiz, 1853. Of the most parsimonious analysis, the tree length generated was 2 529 with a consistency index (CI) of 0.2056 and Homoplasy index (HI) of 0.7944.

The values of uninformative character excluded by CI and HI were 0.1974 and 0.8026 respectively. The retention index (RI) generated from this analysis was 0.6116 and a rescaled consistency index (RC) at 0.1258.

3.5 Phylogenetic relationship among Cichlidae

The topology of the MP tree of cichlid family is shown on Fig. 8. The MP analysis recovered Cichlidae as a monophyletic family with strong support of 1.00 posterior

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probability (pp) of the majority rule consensus tree. Four subfamilies within the Cichlidae were distinctly recovered. Of the four subfamilies, three were recovered monophyletic which included Cichlinae, Etroplinae and Ptychochrominae. On the other hand,

Psuedocrenilabrinae was polyphyletic.

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Fig. 8. Maximum parsimony tree (tree length 2529, CI 0.2056, RI 0.6116) based on mtDNA COI data of some cichlids. Numbers above branches are Bayesian pp > 0.5 and below branches are bs percentages above 50. Bootstrap percentages below 50 are not shown. CI, consistency index; RI, retention index; pp, posterior properbility; bs, bootstrap

The New World Cichlinae (1.00 pp) consisted of four clades: (1) clade I with 0.78 pp

contained a cluster of Herichthys (H. pantosticus, H. tamasopoensis, H. labridens and H.

steindachneri), Acaronia nassa and lapidifer; (2) clade II (0.94 pp) consisted

Chaetobranchus (Chaetobranchus semifasciatus and C. flavescens),

orbicularis and Cichlasoma (Cichlasoma dimerus and C. bimaculatum); (3) clade III (0.96

pp) included sister taxa of brasiliensis and Pterophyllum scalare; (4) clade IV

(0.95 pp) contained a monophyletic clustering of Cichla (Cichla tamensis, C. monoculus,

C. piquiti and C. kelberi). A single branch of Astronotus ocellatus was recovered as the

most basal taxon of the New World cichlids.

The Indian/Madagascar Etroplinae clade (0.94 pp / 0.53 bootstrap (bs) value) consist of

genera Etroplus (Etroplus maculatus and E. suratensis) and Paretroplus (Paretroplus

kieneri and P. damii). This subfamily is recovered as the basal-most of the entire cichlid

family.

The Ptychochrominae which is endemic to Madagascar alone included Oxylapia pollii and

a sister taxa of Ptychochromis inornatus and P. oligacanthus. This group was recovered

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with pp value of 1.00 in between New World Cichlinae and Old World

Pseudocrenilabrinae.

Of the four subfamilies, the mainland African Pseudocrenilabrinae (0.99 pp) was recovered with the largest number of clusters. Excluding the Heterochromis (H. multidens) which was recovered as a sister clade to the clade of New World and African cichlids. However, 11 clades were recovered within the subfamily Pseudocrenilabrinae:

(1) Clade A (1.00 pp / 91 bs) was recovered by the clustering of a non-monophyletic

Serranochromis (S. altus, S. angusticeps, S. robustus, and S. macrocephalus) and a monophyletic Sargochromis (S. carlotte, S. codringtoni and S. giardia). Thoracochromis included T. albolabris and T. buysi were recovered as sister taxa and the basal group within this clade; (2) Clade B (0.99 pp / 53 bs) contained the highest cluster of genera which included sister groups of Metriclima greyshaki and elongates, as well as sister taxa taeniolatus and Trematocranus placodon.

Pseudocrenilabrus philander is recovered the basal taxon within this clade. Clade B shares same ancestor with clade A; (3) Clade C (0.86 pp) consisted of only dickfeldi; (4) Clade D (1.00 pp / 100 bs) contained sister groups of Tilapia guisana and T. sparmanii; (5) clade E (1.00 pp) contained a sister taxa of Coptodon rendallii and C. zillii,

Tilapia guineensis, T. tholloni and also a sister taxa of T. dageti and T. mariae; (6) Clade

F (1.00 pp / 74 bs) contained a non-monophyletic Sarotherodon (S. galilaeus, S. lohbergeri and S. melanotheron) and Tristramella simons. This clade formed a sister clade with Clade E; (7) Clade G (0.91 pp) also recovered a non-monophyletic

Oreochromis included O. macrochir, O. mossambicus, O. urolepis and O. niloticus. The

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Nile tilapia O. niloticus is seen as the most basal taxon among the Oreochromis.

Furthermore, within Clade G, the Magadi tilapia Alcolapia grahami is embedded in this clade; (8) Clade H (1.00 pp / 100 bs) was formed by monotypic genus Steatocranus which consisted S. gibbiceps, S. tinanti and S. casuarius; (9) Clade I (0.99 pp / 62 bs) contained a monotypic genus Etia nguti; (10) Clade J (1.00 pp / 100 bs) was recovered by the clustering of species of Tylochromis. Included in this clade are sister taxa of T. aristoma and T. lateralis, T. bangwelenesis was the sister taxa to T. polylepis. T. pulcher and T. sudanensis were always recovered as individual branches. Moreso, T. sudanensis is always recovered basal-most with this group; (11) Clade K (1.00 pp / 86 bs) consisted sister groups Hemichromis bimaculatus and H. fasciatus as well as sister groups H. letourneuxi and H. saharae.

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Fig. 9. One of the 73 most parsimonious trees (tree length 343, CI 0.6035, RI 0.9377) based on mtDNA COI data of representative of Oreochromini lineage. Numbers above branches are Bayesian pp > 0.5. CI, consistency index; RI, retention index; pp, posterior properbility

3.6 Relationship among some species within the Oreochromini lineage

Number of nucleotide basebairs included in the Oreochromini lineage analysis was 652, of which 30 characters were excluded and 622 characters were included. All characters were of the type “unord” and are equally weighted. Four hundred and forty four characters remained constant throughout this analysis having five parsimoniously uninformative variable characters and 262 variable characters considered as parsimony-informative character in the analysis. Furthermore, gaps were treated as missing and optimisation of character-state was treated with the command prompt DELTRAN (i.e. delayed transformation) in the phylogenetic analysis. Out of 76 included taxa, 73 phylogenetic trees were generated from this maximum parsimony (MP) analysis. Tree length generated was 343 with a consistency index (CI) of 0.6035 and Homoplasy index (HI) of

0.3965. The values of uninformative character excluded by CI and HI were 0.5976 and

0.4024 respectively. The retention index (RI) generated from this analysis was 0.9377 and a rescaled consistency index (RC) at 0.5659.

The MP tree recovered orechromini lineage monophyletic (1.00 pp) in Fig. 9. outgroup used were some species of Tylochromis including T. polylepis, T. lateralis, T. pulcher and

T. sudanensis. Within the Orechromini lineage Tristamella simonis was recovered as the

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most basal species (1.00 pp). Tristamella simonis is a sister taxa to all the Sarotherodon species (S. galileus, S. lohbergeri and S. melanotheron) included in this analysis. The genus Oreochromis was recovered non-monophyletic having Orechromis niloticus as the basal species (Fig. 5). The two populations (FSL and SWL) of Magadi tilapia (Alcolapia grahami) was recovered monophyletic in a major clade which is a sister clade to O. andersonii and O. macrochir. Within the A. grahami clade, three different clustering were observed. These clustering had combination of both populations (Fig. 5).

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4. DISCUSSION

The specimens from which DNA was extracted comprised 10 Alcolapia grahami specimens (five specimens from the Fish Spring Lagoon and five specimens from the

South West Lagoon) and six Thoracochromis albolabris specimens. They gave good DNA quality when visualised on agarose gels. Successful amplification of polymerase chain reaction (PCR) results were achieved from all the 16 reactions following the PCR procedure of Steinke and Hanner (2011). Sequencing success was also accomplished from all the 16 PCR reactions. The dataset which included a total of 143 sequences, 121 mined sequences and 16 sequences generated were aligned, screened and filtered for uniqueness. Further, BRONX analysis was conducted to identify short variant segments and corresponding invariant flanking regions in the dataset (Little 2011).

The concept of barcoding species was proposed to help resolve species identification

(Hebert et al. 2003). However, since the invention of DNA barcoding (Hebert et al. 2003), molecular scientists have not conclusively determined whether barcoding region gives satisfactory information for it to be a perfect tool for species identification. Also, there has not been a universal threshold that could distinguish interspecific variation. However for many taxonomic groups, cytochrome c oxidase subunit I (COI) region has proven effective for some species delimitation (e.g. Ward et al. 2009; Van der Bank et al. 2012).

Yet, COI gene has been ineffective for some other species (Song et al. 2008; Siddall et al. 2009). This has led to controversies over some proposed barcoding applications. In this study, COI’s potential was tested as a good barcoding marker for cichlids. A good barcode application should show greater variation between species (interspecific) than

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within species (intraspecific) (Meyer and Paulay 2005). Among the different ways of testing the presence of barcode gap, two approaches were used in this study. Firstly, the average interspecific distance was compared against the intraspecific distance. It was observed that the distances between species was remarkably greater than that within species. This suggests that COI gene shows the presence of barcode gap (Meyer and

Paulay 2005; Van der Bank et al. 2013). Secondly, as proposed by Meier et al. (2008), the smallest interspecific distance was compared against the greatest intraspecific distance. The outcome also showed existence of barcode gap, thus corroborating the fact that COI gene is a capable DNA region to classify species within cichlid fishes.

Furthermore, this short mtDNA marker (COI) has been proven successful in other animals

(Kochzius et al. 2010; Van der Bank et al. 2012; Čandek and Kunter 2015).

When the COI segment of the mitochondrial DNA was used in this study as a discriminatory tool for species identification, there was variation among the three distance based approaches. From the three approaches evaluated, near neighbour and best close match gave a high performance, with the near neighbour giving the best results in distinguishing species within genus and in the entire dataset. This supports the effiveness of COI gene for identification of cichlids species (Nwani et al. 2011). However, the approach of BOLD identification (BOLD id) criteria performed the poorest amongst the distance based techniques in distinguishing cichlids to species level. BOLD id gave the lowest number of correct identification and had the most sequence with ambiguity as well as highest rate of incorrectly identified sequence to cichlid species. This weak performance of COI applied under the BOLD id has been inferred by Meyer and Paulay

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(2005). These authors suggested the low values of BOLD id criteria could result from the unverified one percent threshold applied in BOLD id identification. Therefore COI gene should not be seen as the consequence of barcoding ineffectiveness based on BOLD id approach.

4.1 Phylogenetic relationships

The maximum parsimony (MP) phylogenetic analysis was in accordance with former phylogenetic analysis of Cichlidae done by McMahan et al. (2013), with some notable differences. There is a false sister relationship between Ptychochrominae lineage and the

African Psuedocrenilabrinae lineage which is most likely caused by random error. This random error occurs because the barcoding gene (652 bp) does not provide enough information. However, this random error could be prevented by adding more genes especially nuclear genes to the sample in future.

The cichlid family was shown to be a monophyletic family with strong support (Bayesian inference posterior probability is 1.00). The monophyly of Cichlidae that resulted from a single barcoding gene analysis is in concordance with previously generated phylogenetic topologies of cichlid family from combined genes (e.g. Farias et al. 2000; McMahan et al.

2013). In this study, the position of the Hemichromis as the most basal genus within the

African clade conforms to earlier works of Stiassny (1991) and Van der Bank (1994) based on morphological and molecular grounds respectively. Here, Etia nguti was recovered as a monotypic species. Also E. nguti was observed as a sister group to clade

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A to H. This is in concordance with previous phylogenetic works (Schliewen and Stiassny

2003; Schwarzer et al. 2009; Dunz and Schliewen 2013).

Clades F and G contains species that grouped just as the Oreochromini tribe as recorded by Dunz and Schliewen (2013). Specifically, looking at the different populations of

Alcolapia grahami within the Oreochromini tribe, DNA barcoding gene cannot tell the two populations apart unlike the microsatellite genes used in Kavembe et al. (2014). This is because DNA barcoding gene COI was developed for fast identification purposes

(Herbert et al. 2003). From the physical distance between Fish Springs Lagoon and South

West Lagoon of 18 km, the only fish species found in these lagoons is A. grahami.

However, these lagoons are a long distance apart such that their waters could not have mixed. Therefore, there could not have been exchange of gene pools of fauna between these lagoons in the recent times. Therefore, it is plausible that the A. grahami population in the FSL might have developed a different gene pool compared to the population found in the SWL but COI could not tell them apart. Maybe the addition of some nuclear genes and or microsatellite genes for population genetics could improve the results and answer the questions whether the fish in different lagoons of Lake Magadi have speciated.

However, the positions of base pair where variations occurred within the two populations of A. grahami sampled is shown on Appendix 3. Hypothetically, the observed topology might be as a result of exchanged gene pools between the fish populations of the FSL and the SWL. This might have resulted from the dispersal of A. grahami eggs through the prevalent predators e.g. the flamingos which frequent Lake Magadi ecosystem in large

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numbers. These birds could have transferred the eggs as they shuffle between lagoons for food.

However, there are some inconsistencies with the phylogenetic aspect of this study compared to previous studies. These discrepancies are mostly due to molecular techniques used. For example a single gene (COI) could not support the findings from the 24 gene phylogeny in resolving the phylogenetic position of Heterochromis multidens from (Keck and Hulsey 2014). In future, to prevent such inconsistences more genes especially the nuclear genes should be added to the single mitochondrial gene used in this study.

In this study, the omnivorous (Van der Bank, 1994) within the

Oreochromini tribe that includes O. andersonii, O. macrochir, O. mossambicus and O. niloticus were grouped contrarily to previous work (Van der Bank, 1994; Kavembe et al.

2014). This is due to the fact that COI gene of the mitochondrial DNA alone was used.

Based on allozyme data from 40 protein coding loci, six different phylogenetic trees were reconstructed by Van der Bank (1994) and all trees placed O. mossambicus and O. andersonii as sister taxa. In the six phylogenetic trees, O. macrochir was recovered basally to O. andersonii and O. mossambicus (Van der Bank, 1994). Also, using microsatellite and mitochondrial genes that included control region and ND2, O. andersonii and O. mossambicus were recovered as sister taxa with a 99% bootstrap value whereas O. macrochir was basally placed as a sister taxon to both O. andersonii and O. mossambicus (Kavembe et al. 2014). Contrary to previous works, this present

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study gave a completely different result. Here, Oreochromis mossambicus was recovered as the basal species to Alcolapia grahami, and both O. andersonii and O. macrochir were sister taxa (Fig. 8).

The species composition in Clade A are in concordance with the findings of Joyce et al.

(2005) and Musilová et al. (2013) where Serranochromine lineage was shown to be monophyletic. Thoracochromis is a genus within the Serranochromines lineage and all species of this genus including T. albolabris and T. buysi portrayed a monopyletic sister relationship.

Within the New World cichlids, Clade I contains different tribes such as

(Herichthys), Cichlasomatini (Acaronia), and Retroculini (Retroculus). Retroculini lineage that was recovered in this study as the sister lineage to the Heroini and Cichlasomatini tribes is not in accordance with the result of Smith et al. (2008) and McMahan et al. (2013) who based their analyses on eight (morphology, COI, Cyt b, NADH4, H3, Tmo-4c4, Tmo- m27 and S7) and four (16S, COI, Tmo and H3) characters and genes respectively. In the present study (Fig. 8) Chaetobranchus and Chaetobranchopsis (representatives of the

Chaetobranchini tribe) formed sister taxa with Cichlasoma dimerus and C. bimaculatum

(species within the Cichlasomatini tribe). This is in discordance with the results of Smith et al. (2008). Smith et al. (2008) determined that the Cichlasomatini is more closely related to the Geophagini tribe. Furthermore, Pterophyllum scalere, which is a species belonging to the large group of Heroini lineage, was recovered in this study (Fig. 8) as the sister taxon of Geophagus brasiliensis, a species of the Geophagini tribe. The relationships

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between these species are in disagreement with the findings of Smith et al. (2008), where it was reported that Heroini was shown to be the sister lineage to Cichlasomatini. The final clade containing Cichla and Astronotus, which belong to tribes Cichlini and

Astronotini respectively, is also in conflict with the studies of Smith et al. (2008) and

McMahan et al. (2013). Here, species belonging to the Heroini and Cichlasomatini tribes were determined to be polyphyletic. This is in disagreement with Smith et al. (2008) and

McMahan et al. (2013).

In this study, Ptychochrominae and Etroplinae are not discussed because they are not the main focus of this work. Some representatives within these two subfamilies were included just to show a reflection of the four subfamilies in Cichlidae.

In this present age of molecular biology, DNA barcoding has been a very useful techniques for species identification. However, some methods comparable to DNA barcoding have been employed to resolve the identification of some species where morphological methods have proved inadequate (e.g. Zettler et al. 2002; Lewis and Lewis

2005; Abriouel et al. 2008; Mitchell 2008; Pagès et al. 2009). For these species, it could have been helpful to use DNA barcoding gene which is a standard segment to aid identification. Out of the numerous merits of barcoding, the fact that the application of

DNA barcoding in identification of species can be achieved without the assistance of an experienced taxonomist in some cases remains impressive. DNA barcoding also serves as a quality control measure to corroborate species identification, even from dried snake venom (Pook and McEwing 2005). Commercial applications of barcoding in invasive

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species detection and fishery management was proposed by Rock et al. (2008) and Ward

(2009).

Some natural occurrences that may possibly hamper barcoding efficiency are error in species identification, heteroplasmy, introgression, incomplete sorting, paternal leakage, polyploidization and recent speciation (Hebert et al. 2003; Mitchell 2008; Ward 2009;

Rubinoff 2006; Rock et al. 2008; Langhoff et al. 2009). Depending on the acquired data these events are recorded to happen at different levels. An additional possible drawback is that the barcoding region may be less informative to properly distinguish between and within species. According to the results established by Hebert et al. (2003), Meier et al.

(2006), Mitchell et al. (2008) and Rock et al. (2008) not all species show variations in their

DNA standard segment. Furthermore, there is concurrence between researchers that

DNA standard segment includes less information to substantially uphold phylogenetic facts (Hebert et al. 2003; Meier et al 2006; Pagès et al. 2009).

In summary, DNA barcoding has its limitations and many researchers employing the technique should recognise its limitations. Nonetheless, old-fashioned established on morphological analysis has also got its own limitations (Hebert et al. 2003;

Rubinoff 2006; Packer et al. 2009). In recognition of this, Ribak (2010, page 28) stated that “it is naïve to hope for a system of identification that can identify all species without making any errors”.

Page | 66

5. CONCLUSION

The current study provides for the first time COI sequences of Alcolapia grahami,

Thoracochromis albolabris and T. buysi. These sequences have been deposited in

BOLDsystems.org. In addition, DNA barcoding was able to identify correctly species within the cichlids family using the near neighbour and best close match parameters.

Therefore, DNA barcoding is recommended for the identification of cichlids fishes such as A. grahami, T. albolabris and T. buysi as well as other cichlids within the family

Cichlidae. However, the BOLD identification criteria performed poorly in identification of

T. albolabris and the entire data set. This might be due to the universal one percent threshold that may be subject to review in future studies.

The DNA barcode had smaller amount of sequence basepairs (652) and lesser parsimony informative characters (262) when compared to Sparks and Smith (2004) dataset which included multiple evidences (2 222 bp, 842 parsimony informative characters). Since there was fewer information presented in this study, it was anticipated that mtDNA COI gene would have greater problems than nucleotide data from multiple evidences

(mtDNAs and nuclear genes) of Spark and Smith (2004), Spark (2008), Dunz and

Schliewen (2013) and McMahan et al. (2013).

Single mtDNA COI has problems in establishing deeper level relationships within the

African Pseudocrenilabrinae cichlids, particularly the genus Heterochromis from Central

Africa as mentioned before. For this reason, COI could not corroborate the fact that

African Pseudocrenilabrinae was a monophyletic subfamily. Also, it could not resolve the

Page | 67

monophyly of Oreochromini tribe: Sarotherondon and Tritramella were recovered as sister taxa to the Coptodoni tribe instead of grouping with the Oreochromis and Alcolapia clade. Furthermore, COI could not distinctly differentiate between populations of Alcolapia grahami from the Fish Spring and South Western lagoons of Lake Magadi. In general mtDNA of COI alone showed that it cannot successfully control the complex species relationships existing within the family Cichlidae. However, COI distinguished appropriately the African Pseudocrenilabrinae from the New World Cichlinae. It also confirmed the position of A. grahami within the Oreochromini tribe and within the

Serronochromine tribe it confirmed the positions of Thoracochromis albolabris and T. buysi with strong support.

However, more samples of cichlids should be added and morphological as well as other molecular markers such as nuclear genes are recommended in future studies. It may yield a more comprehensive phylogenetic result. Nevertheless, the result of this study contributed to the global information on genetic biodiversity and species identification of some African cichlids such as A. grahami, Thoracochromis albolabris and T. buysi. It is also the very first report of DNA barcoding of Tilapia guinasana (an endangered species, see Skelton (2001)). These will provide further information that can help in fish aquaculture and fish product authentication. All four (A. grahami, the two Thoracochromis spp. and Tilapia guinasana) species have extremely limited natural distributions and are, therefore, only accessible to very few people in the world.

Page | 68

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APPENDIX 1. Cichlid Authorities

Scientific name Authority and Year Acaronia nassa (Heckel, 1840)

Alcolapia grahami (Seegers and Tichy 1999) burtoni (Günther 1894) Astatotilapia desfontainii (Lacépède 1802)

Astronotus ocellatus (Agassiz 1831) Aulonocara jacobfreiberghi (Johnson, 1974) Aulonocara nyassae Regan 1922 Aulonocara stuartgrandti Meyer & Riehl, 1985 Boulengerochromis microlepis (Boulenger 1899) Chaetobranchopsis orbicularis (Steindachner, 1875) Chaetobranchus flavescens Heckel 1840 Chaetobranchus semifasciatus Steindachner,1875 welwitschi (Boulenger 1898) Cichla kelberi Kullander & Ferreira, 2006 Cichla monoculus Agassiz, 1831 Cichla piquiti Kullander & Ferreira, 2006 Cichla temensis Humboldt, 1821 Cichlasoma bimaculatum (Linnaeus, 1758) Cichlasoma dimerus (Heckel, 1840)

Cichlasoma nigrofasciatum (Günther 1867) Cichlasoma ornatum Regan 1905

Coptodon rendalli (Boulenger, 1897)

Coptodon zillii (Gervais, 1848) Eretmodus cyanostictus Boulenger 1898 Etia nguti Schliewen & Stiassny, 2003 Etroplus maculatus (Bloch, 1795) Etroplus suratensis (Bloch, 1790) Trewavas 1935 Geophagus brasiliensis (Quoy & Gaimard, 1824)

Geophagus harreri Gosse, 1976 Hemichromis bimaculatus Gill 1862

Hemichromis fasciatus Peters 1857 Hemichromis letourneuxi Sauvage, 1880 Hemichromis saharae Sauvage, 1880

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Herichthys labridens (Pellegrin, 1903) Herichthys pantostictus (Taylor & Miller, 1983) Herichthys steindachneri (Jordan & Snyder, 1899) Herichthys tamasopoensis Artigas Azas, 1993 Heterochromis multidens (Pellegrin 1900)

Julidochromis dickfeldi Staeck, 1975 Labidochromis caeruleus Fryer, 1956 labidochromis fuelleborni Trewavas, 1935 Labidochromis vellicans Trewavas 1935 Lamprologus congoensis Schilthuis 1891 elongatus (Boulenger 1898) Lipochromis obesus (Boulenger 1906) Metriaclima greshakei Meyer & Förster, 1984) Metriaclima lanisticola (Burgess 1976) Nanochromis nudiceps (Boulenger 1899) Neolamprolagus brevis (Boulenger 1899)

Oreochromis andersonii (Castelnau, 1861) Oreochromis macrochir (Boulenger, 1912)

Oreochromis mossambicus (Peters, 1852) Oreochromis mossambicus (Peters, 1852)

Oreochromis niloticus (Linnaeus 1758) Oreochromis urolepis (Trewavas, 1966) Orthochromis polyacanthus (Boulenger 1899) Oxylapia polli Kiener & Maugé, 1966 Paretroplus damii Bleeker, 1868

Paretroplus kieneri Arnoult, 1960 Boulenger 1898 Petrochromis tanganicae (Günther 1894) Pharyngochromis acuticeps (Steindachner 1866) milomo Oliver, 1989 (Trewavas, 1935) Pseudocrenilabrus philander (Weber 1897) Pseudotropheus elongatus Fryer, 1956 Pterophyllum scalare (Schultze 1823) Ptychochromis inornatus Sparks, 2002 Ptychochromis oligacanthus (Bleeker, 1868) (Castelnau, 1855)

Sargochromis carlottae (Boulenger, 1905) Sargochromis codringtoni (Boulenger, 1908)

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Sargochromis giardi (Pellegrin, 1903) Sarotherodon Rüppell, 1852 Sarotherodon galilaeus Linnaeus, 1758 Sarotherodon lohbergeri (Holly, 1930) Sarotherodon melanotheron Rüppell, 1852 Serranochromis altus Winemiller & Kelso-Winemiller, 1991 Serranochromis angustiuceps (Boulenger 1907) Serranochromis macrocephalus (Boulenger, 1899) Serranochromis robustus (Günther, 1864) Steatocranus casuarius Poll, 1939 Steatocranus gibbiceps Boulenger 1899 Steatocranus tinanti (Poll, 1939) Teleogramma gracile Boulenger 1899 Thys van den Audenaerde and Trewavas Thoracochromis albolabris 1969 Thoracochromis buysi (Penrith, 1970)

Thorichthys meeki Brind 1918 Tilapia dageti Thys van den Audenaerde, 1971 Tilapia guinasana Trewavas, 1936 Tilapia guineensis (Günther 1862) Tilapia mariae Boulenger 1899 Tilapia sparrmanii Smith, 1840 Tilapia tholloni (Sauvage, 1884)

Trematocranus placodon (Regan 1922) Tristramella simonis (Günther, 1864) Tropheus morri Boulenger 1898 Tylochromis aristoma Stiassny, 1989 Tylochromis bangwelensis Regan, 1920 Tylochromis lateralis (Boulenger, 1898) (Boulenger, 1900) Tylochromis pulcher Stiassny, 1989 Tylochromis sudanensis Daget, 1954 Abudefduf saxatilis (Linnaeus, 1758) Embiotoca jacksoni Agassiz, 1853

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2. The distribution of cichlids used in this study

SPECIES DISTRIBUTION Acaronia nassa (Heckel, Found in the Amazon, Orinoco and other Basins in 1840) northern. Alcolapia grahami Lake Magadi Kenya. (Boulenger, 1912) Astronotus ocellatus Amazon River Basin in Peru, Colombia and Brazil; (Agassiz 1831) French Guiana. Reported from Argentina Chaetobranchopsis Amazon River Basin, along the Amazon River from the orbicularis (Steindachner, mouth or the Negro River to Marajó Island, and in 1875) Amapá, Brazil. Chaetobranchus Amazon River Basin, in Peru and Brazil; Orinoco River flavescens Heckel 1840 Basin in Venezuela (Rio Apure); rivers of Guyana, Suriname, French Guiana, and Amapá (Brazil). Chaetobranchus Amazon River Basin, along the Amazon-Solimões River semifasciatus from Tabatinga to Óbidos, Brazil. Steindachner,1875 Cichla kelberi Brazil. Kullander & Ferreira, 2006 Cichla monoculus Rio Solimões-Amazonas along the main channel and Agassiz, 1831 lower courses of tributaries; Peru, Colombia and Brazil; including Araguari and lower Oyapock rivers north of the Amazon. Probably much more widespread in the lowland . Cichla piquiti Brazil. Kullander and Ferreira, 20 06 Cichla temensis Amazon River Basin in the Negro and Uatumã River Humboldt, 1821 drainages; Orinoco River Basin in tributaries of the Orinoco River in Venezuela and Colombia. Cichlasoma bimaculatum Orinoco River Basin, River Venezuela; Guianas, from the (Linnaeus, 1758) Essequibo River to the Sinnamary River; Amazon River Basin, in the upper Branco River Basin. Cichlasoma dimerus Paraná River Basin, in the Paraguay River drainage in (Heckel, 1840) Brazil, Bolivia and Paraguay, and the Paraná River drainage of Argentina.

Coptodon rendalli Kasai Drainage (middle Congo River Basin), throughout (Boulenger, 1897) upper Congo River drainage, Lake Tanganyika, Lake Malawi, Zambesi, coastal areas from Zambesi Delta to Natal, Okavango and Kunene. Also in the Limpopo Province. Coptodon zillii (Gervais, South Morocco, , Niger-Benue System, rivers 1848) Senegal, Sassandra, Bandama, Boubo, Mé, Comoé, Bia,

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Ogun and Oshun, Volta System, Chad-Shari System, Middle Congo River Basin in the Ubangi, Uele, Ituri and Itimbiri (Democratic ), Lakes Albert and Turkana, system and the Jordan system. Etia nguti Schliewen & Only known from the type localities in the region of Nguti Stiassny, 2003 in the Mamfue River, a tributary of the upper Cross River, Cameroon. Etroplus maculatus India and Sri Lanka. (Bloch, 1795) Etroplus suratensis India and Sri Lanka. (Bloch, 1790) Geophagus brasiliensis Coastal drainages of eastern and southern Brazil and (Quoy & Gaimard, 1824) Uruguay. Hemichromis bimaculatus Widely distributed in , where it is known from Gill 1862 most hydrographic basins, associated with forested biotopes. Also reported from coastal basins of Cameroon, Democratic Republic of the Congo and Nile Basin, but at least its presence in Cameroon is unconfirmed, limits this species to , Sierra Leone and Liberia. Species boundaries in the genus Hemichromis remain unclear. Hemichromis fasciatus Widely distributed in West Africa, where it is known from Peters 1857 most hydrographic basins. Also in the Nile Basin, Lake and in the Upper Zambezi. Distribution of this species and overlap with Hemichromis elongatus unclear, but probably absent from the Congo Basin. Introduced around 1970 in a stream fed by hot springs in Villach (Austria). Hemichromis letourneuxi Nile to Senegal and from to Côte d'Ivoire. Sauvage, 1880 Hemichromis saharae Nile to Senegal and from North Africa to Côte d'Ivoire. Sauvage, 1880 Herichthys labridens Atlantic slope, in the Panuco River Basin, Mexico. (Pellegrin, 1903) Herichthys pantostictus Atlantic slope, in the Panuco River Basin, Mexico. (Taylor & Miller, 1983) Herichthys steindachneri Atlantic slope of . Endemic to the (Jordan & Snyder, 1899) Tamasopo, Gallinas and Ojo Frio rivers in the Panuco River Basin. Herichthys tamasopoensis Atlantic slope, in the Tamasopo River of Panuco River Artigas Azas, 1993 Basin, Mexico. Heterochromis multidens Middle Congo River Basin, including the rivers Ubangi, (Pellegrin 1900) Ngupaya, Tshuapa, Dja and Sanga. Julidochromis dickfeldi Endemic to the south western part of Lake Tanganyika, Staeck, 1975 north of Sumbu national park in Zambia.

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Metriaclima greyshaki Endemic to Makokola in the southeast arm of Lake (Meyer & Förster, 1984) Malawi. Oreochromis andersonii Ngami Basin, Okavango River; Kunene River and (Castelnau, 1861) Mossamedes, Angola; upper Zambezi, Kafue River; middle Zambezi, Lake Kariba and Cabora Bassa since construction of dams. Oreochromis macrochir Kafue, Upper Zambezi, and Congo River systems; (Boulenger, 1912) introduced elsewhere in Africa and in Hawaiian Islands Also in the Okavango and Ngami region, Kunene Basin, Chambezi and Bangweulu region. Oreochromis Lower Zambezi, Lower Shiré and coastal plains from mossambicus (Peters, Zambezi Delta to Algoa Bay. Occurs southwards to the 1852) Brak River in the eastern Cape and in the Transvaal in the Limpopo System. Widely introduced for aquaculture, but escaped and established itself in the wild in many countries, often outcompeting local species. Oreochromis niloticus Naturally occurring in coastal rivers of , Nile Basin (Linnaeus 1758) (including lake Albert, Edward and Tana), Jebel Marra, Lake Kivu, Lake Tanganyika, Awash River, various Ethiopian lakes, Omo River system, Lake Turkana, Suguta River and Lake Baringo. In West Africa natural distribution covers the basins of the Senegal, Gambia, Volta, Niger, Benue and Chad, with introduced specimens reported from various coastal basins. Oreochromis urolepis Rufigi River and its tributaries; the Kilombero and Great (Trewavas, 1966) Ruaha rivers, but not in the delta; the Kingani, Mbenkuru and Wami rivers, all in Tanzania. Oxylapia polli Kiener & Marolambo rapids, , Madagascar. Maugé, 1966 Paretroplus Endemic to the northwestern part of Madagascar. damii Bleeker, 1868 Paretroplus kieneri Endemic to Kamoro area (north of Madagascar). Arnoult, 1960 Protomelas taeniolatus Endemic to Lake Malawi where it is widely distributed. (Trewavas, 1935) Pseudocrenilabrus From the Orange River System and southern Natal philander (Weber 1897) northwards throughout southern Africa, extending to southern Congo Basin tributaries and Lake Malawi. Pseudotropheus Endemic to Lake Malawi. Occurs in Nkata Bay and elongates Fryer, 1956 Mbamba Bay. Pterophyllum scalare Amazon River Basin, in Peru, Colombia, and Brazil, (Schultze 1823) along the Ucayali, Solimões and Amazon rivers; rivers of Amapá (Brazil), Rio Oyapock in French Guiana; Essequibo River in Guyana. Ptychochromis inornatus Madagascar. Sparks, 2002

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Ptychochromis Endemic to the coasts of Madagascar, except the central oligacanthus (Bleeker, western coast and Nosy-Be Island. 1868) Retroculus lapidifer Amazon River Basin, in the Tocantins and Capim River (Castelnau, 1855) Basins, Brazil. Sargochromis carlottae Okavango River, Upper Zambezi River, Lake Kariba and (Boulenger, 1905) the Kafue River (Angola, Namibia, Botswana, Zambia and Zimbabwe). Sargochromis codringtonii Okovango River System, Upper and Middle Zambezi (Boulenger, 1908) River systems, Kafue River (Angola, Namibia, Botswana, Zambia and Zimbabwe). Sargochromis giardia Kunene River System, Okavango River, Upper and (Pellegrin, 1903) Middle Zambezi rivers, Kafue River (Angola, Namibia, Botswana, Zambia and Zimbabwe). Sarotherodon galilaeus Jordan System, especially in lakes; coastal rivers of Linnaeus 1758 Israel; Nile system, including the Delta lakes and Lake Albert and Turkana; in the Congo Basin in the Lower and Middle Congo River from to , and in the Lower Kasai; in West Africa in the Senegal, Gambia, Casamance, Géba, Konkouré, Sassandra, Bandama, Comoé, Niger, Volta, Tano, Lake Bosumtwi, Mono, Ouémé, Ogun, Cross, Benue, Logone, Shari and Lake Chad; Draa (Morocco), Adrar (Mauritania); Saharian oases Borku, Ennedi and Tibesti in northern Chad. Sarotherodon lohbergeri Lake Barombi Mbo, a tributary of Kake River (affluent of (Holly, 1930) Lake Barombi Mbo) and Kumba stream (tributary of the outlet of Barombi Mbo), west Cameroon. Sarotherodon Lagoons and estuaries from Mauritania to Cameroon. melanotheron Rüppell, Introduced to several countries in Asia, USA and . 1852 Serranochromis altus Upper Zambezi River, Zambia and Okavango Drainage Winemiller & Kelso- in Botswana and Namibia. Winemiller, 1991 Serranochromis Kunene River System (Angola and Namibia), Okavango angusticeps (Boulenger River, Upper Zambezi, and Kafue Rivers (Angola, 1907) Namibia, Botswana, Zambia, Zimbabwe), and Luapula- Moeru (Congo River system) in Democratic Republic of the Congo and Zambia. Serranochromis Angola, Botswana, Namibia, Zambia, Zimbabwe and macrocephalus Democratic Republic of the Congo in the Kunene River (Boulenger, 1899) System (Angola and Namibia); Okovango River and swamps; Upper Zambezi System including Lake Cameia, Kafue River and Luangwa System; Luapula- Mweru, Lulua River and Angolan Kasai (Upper and Middle Congo River Basin).

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Serranochromis robustus Lake Malawi, rivers flowing into it and the Upper Shire (Günther, 1864) River. Translocated to the upper Ruo River in Malawi and also to Swaziland. Steatocranus casuarius Pool Malebo and the Lower Congo River in both Poll, 1939 Republic of Congo and Democratic Republic of the Congo. Steatocranus gibbiceps Pool Malebo and the Lower Congo River in Republic of Boulenger 1899 Congo and Democratic Republic of the Congo Steatocranus tinanti (Poll, Pool Malebo and the Lower Congo River in Republic of 1939) Congo and Democratic Republic of the Congo. Thoracochromis Kunene River in Angola and Namibia. albolabris Thys van den Audenaerde and Trewavas 1969 Thoracochromis buysi Kunene River in Angola and Namibia. (Penrith, 1970) Tilapia dageti Thys van Upper Senegal, Upper and Middle Niger systems, Upper den Audenaerde, 1971 Comoe, Volta, Mono, Bénoué and Lake Chad. Tilapia guinasana Endemic to Lake Guinas, Namibia. Introduced to Lake Trewavas, 1936 Otjikoto and several reservoirs in Namibia. Also occurs in subterranean rivers. Tilapia guineensis Coastal Basins, fresh waters, brackish and marine (Günther 1862) waters from mouth of Senegal River (Senegal) to mouth of the Cuanza River (Angola), sometimes ascending far up rivers. Tilapia mariae Boulenger Coastal lagoons and lower river courses from the Tabou 1899 River (Côte d'Ivoire) to the Kribi River (Cameroon), but absent from the area between the Pra River (Ghana) and Benin. Also recorded from the lower Ntem, Cameroon (81260). Tilapia sparrmanii Smith, Kasai Drainage including the Lulua and Kwango (middle 1840 Congo River Basin), Upper Congo River Basin including the Upper Lualaba, Luvua, Lake Mweru, Luapula, Lufira and Upemba region, Upper Cuanza, Kunene, Okavango, Lake Ngami, Zambezi, Limpopo, Sabi, Lundi, northern tributaries of the Orange River, Lake Malawi and Bangweulu. Tilapia tholloni (Sauvage, From the Ogooué River in Gabon to the Lower Congo 1884) River in Democratic Republic of the Congo, including the Nyanza in Gabon, Kouilou-Niari in Republic of Congo, and the Chiloango in Angola and Democratic Republic of the Congo. Trematocranus placodon Widespread in Lake Malawi, upper Shire River and Lake (Regan 1922) Malombe. Tristramella simonis Lakes Tiberias and Muzairib in the Jordan System. (Günther, 1864)

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Tylochromis aristoma Tshuapa Drainage (Middle Congo River Basin) in Stiassny, 1989 Democratic Republic of the Congo. Tylochromis bangwelensis Chambesi, Upper Luapula and Lake Bangwelu and Regan, 1920 surrounding swamps (Upper Congo River Basin) in Democratic Republic of the Congo and Zambia. Tylochromis lateralis Lower Congo River just below Pool Malebo (Stanley (Boulenger, 1898) Pool) and throughout the Middle Congo River Basin including Pool Malebo, Kasai Drainage, lakes Mai- Ndombe and Tumba, and the rivers Tshuapa, Sangha and Aruwimi but excluding the Ubangi System. Tylochromis polylepis Endemic to Lake Tanganyika. In the Lukuga River (Lake (Boulenger, 1900) Tanganyika outflow), known up to Niemba Tylochromis pulcher Mainstream of the Middle Congo River, lakes Mai- Stiassny, 1989 Ndombe and Tumba, the Kasai River and the Tshuapa River (Middle Congo River Basin) in Democratic Republic of the Congo. Tylochromis sudanensis Upper and Middle Niger, and the rivers Bénoué, Cross Daget, 1954 and Warri in Nigeria and Cameroon. Abudefduf saxatilis Canada to Rhode Island, USA to Uruguay in the western (Linnaeus, 1758) Atlantic, abundant on reefs; around islands of the mid-Atlantic, Cape Verde, and along the tropical coast of western Africa south to Angola. This species is strictly an Atlantic species. Embiotoca jacksoni Fort Bragg in northern California, USA to central Baja Agassiz, 1853 California in Mexico, including Guadalupe Island (off northern central Baja California).

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3. The position of base pair where variations occurred within the two populations of Alcolapia grahami sampled from the Fish Springs Lagoon (FSL) and the South West Lagoon (SWL) as indicated by the bold process numbers below

Species Position of base pair 3164328 460 568

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