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Preliminary insight into the age and origin of the Labeobarbus fish species flockfrom Lake Tana (Ethiopia) using the mtDNA cytochrome b gene

Martin de Graaf a,c,*, Hendrik-Jan Megens b, Johannis Samallo a, Ferdinand Sibbing a

a Experimental Zoology Group, Wageningen Institute of Animal Sciences (WIAS), Wageningen University, Marijkeweg 40, 6700 AH Wageningen, The Netherlandsb Animal Breeding and Genomics Centre, Wageningen Institute of Animal Sciences (WIAS), Wageningen University, Marijkeweg 40, 6700 AH Wageningen, The Netherlandsc Wageningen IMARES, P.O. Box 68, 1970 AB Ijmuiden, The Netherlands

a r t i c l e i n f o

Article history:Received 16 January 2009Revised 22 October 2009Accepted 26 October 2009Available online 28 October 2009

Keywords:CyprinidaeSympatric speciationAdaptive radiationmtDNA sequencesEvolution

a b s t r a c t

The high diversity of Cyprinid fish in Ethiopia’s Lake Tana appears to be an example of ecological differ-entiation and assortative mating leading to rapid sympatric speciation. Lake Tana’s Labeobarbus speciesflock consists of 15 morphological and ecological distinct species. This is the first attempt to determinethe age and origin and inter-species relationships of Lake Tana’s Labeobarbus species using the mtDNAcytochrome b gene. Analysis of cytchrome b sequences shows that Lake Tana’s species flock appears tobe young but the present dataset did not unequivocally support monophyly of Lake Tana’s species. Addi-tional markers are needed to determine whether Lake Tana’s labeobarbs originated from a single or mul-tiple incursion(s) of ancestral L. intermedius in the Lake Tana drainage basin, or the disruption of anancient continuous riverine population by the emergence of the Tissisat waterfalls. Adaptive radiationand speciation within Lake Tana’s Labeobarbus species flock may have occurred in the last 10,000–25,000 years, following the desiccation of Lake Tana around 17,000 years ago, at the same time as LakeVictoria, however, obtaining more data using other (nuclear) markers is urgently required.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

The highly diverse cichlid species flocks in the East AfricanGreat Lakes (Lake Victoria > 500 species, Seehausen, 1996; LakeMalawi > 800 species, Snoeks, 2000; Lake Tanganyika > 250 spe-cies, Snoeks, 2000) are famous examples of explosive adaptiveradiations and rapid speciation events (Fryer and Iles, 1972; Keen-leyside, 1991; Verheyen et al., 2003). Studying adaptive radiationsin such relatively young communities like African cichlid speciesflocks has proven useful in understanding the underlying mecha-nisms of speciation and diversity since phenotypic differences be-tween groups are probably linked directly to the forces drivingspeciation (Rüber et al., 1999; Danley and Kocher, 2001).

Not as well known, but not less spectacular, is the high diversityof cyprinid fishes in Lake Tana, a large lake (3050 km2) situated atan altitude of 1830 m in the north-western highlands of Ethiopia.Lake Tana is the source of the Blue Nile. High waterfalls (40 m)at Tissisat (‘smoking waters’), 30 km downstream from the BlueNile outflow, effectively isolate the lake’s ichthyofauna from thelower Nile basin. Despite the overwhelming abundance (>2000species; Nelson, 1994) of cyprinid fishes throughout the world’sfresh water systems, the Labeobarbus species of Lake Tana (Fig. 1)

form, as far as we know, the only remaining intact species flockof large cyprinid fishes, since the one in Lake Lanao in the Philip-pines, has practically disappeared due to human activities (Korn-field and Carpenter, 1984).

Lake Tana’s Labeobarbus species flock offers a unique opportu-nity to study the selective forces driving diversification and speci-ation in cyprinids (de Graaf et al., 2008). Lake Tana’s Labeobarbusspecies flock is intact, evolved in a geographically isolated system,and is small enough (15 species) to study all components simulta-neously. The adaptive radiation into 15 large (max. 100 cm FL),hexaploid (Golubtsov and Krysanov, 1993) endemic Labeobarbusspecies (Nagelkerke et al., 1994; Nagelkerke and Sibbing, 2000)displayed their potential for extensive trophic diversification. Thisincludes the for cyprinids unexpected specialization for piscivory(Sibbing and Nagelkerke, 2001; de Graaf et al., 2008).

Lake Tana’s species flock is hypothesized to have radiated froman ancestral riverine benthivorous labeobarb species resemblingthe L. intermedius complex that is common in the Ethiopian high-lands (Banister, 1973) and is still present along the shores of LakeTana (Nagelkerke et al., 1994; Nagelkerke and Sibbing, 1998; Sib-bing et al., 1998; de Graaf et al., 2008).

The age and origin of the assumed monophyletic (Nagelkerkeand Sibbing, 1998) Labeobarbus species flock in Lake Tana is un-known. Estimates for the age of Lake Tana range from as recentas 10,000 BP (Grabham and Black, 1925) to as old as 2 Ma(early Pleistocene, Mohr, 1962) and 6 Ma years ago (late Miocene,

1055-7903/$ - see front matter � 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2009.10.029

* Corresponding author. Address: Wageningen IMARES, P.O. Box 68, 1970 ABIjmuiden, The Netherlands. Fax: +31 317 487326.

E-mail address: martin.degraaf@wur.nl (M. de Graaf).

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Chorowicz et al., 1998 and references therein). Cytochrome b(mitochondrial DNA [mtDNA]) has been widely used to addressphylogenetic (e.g. Tsigenopoulos and Berrebi, 2000; Tsigenopouloset al., 2002) and phylogeographic questions (e.g. Kotlík and Berrebi,2001) in the genus (Labeo)Barbus. In this paper we describe the firstattempt to discern historical patterns in the labeobarbs of LakeTana using mtDNA. Specifically, we try to gain insight into a num-ber of prominent questions regarding the evolution of Lake Tana’scyprinids: (1) determine the age and origin of Lake Tana’s Labeo-barbus species, (2) whether Lake Tana’s species flock forms amonophyletic group, (3) the level of genetic divergence amongEthiopian Labeobarbs and (4) whether historical relationshipsamong species of Lake Tana’s species flock can be determined.

2. Materials and methods

2.1. Collection and isolation of DNA

Labeobarbus species were collected in Lake Tana and several riv-ers and lakes of different drainage systems during 1999–2001(Fig. 2). Pieces of the dorsal fin from collected specimens were pre-

served in 70% ethanol and DNA was extracted following the Tech-nical manual Wizard Genomic DNA Purification Kit of Promega.Labeobarbs from Sago River (tributary to Lake Chamo; 1 specimen),Arer River (1 specimen), Bulbula River (1 specimen), Lugo River(tributary to Didessa River; 3 specimens) and Dabena River (tribu-tary to Didessa River; 1 specimen) were collected by Abebe Geta-hun (Addis Ababa University, Ethiopia) and the sequences wereprovided to us by William Jordan (British Museum of Natural His-tory, London, UK).

2.2. Polymerase chain reaction, amplification and sequencing

For PCR amplification we used primers L15267 (50-AAT GAC TTGAAG AAC CAC CGT-30) and H16461 (50-CTT CGG ATT ACA AGA CC-30) (Briolay et al., 1998). Numbers refer to the position of the 30 endof the primers in the complete mitochondrial DNA sequence of thecarp Cyprinus carpio (GenBank Accession No. X61010). The use ofL15267 and H16461 in the PCR reactions resulted in the amplifica-tion of a complete of cytochrome b gene (in total 1140 basepairs(bp)). The volume of each PCR was 50 ll and consisted of 1.8 mMMgCl2 (Promega), 1 lM of each primer, 1 ll of 10 mM dNTP-mix

Fig. 1. Heads of the 15 Labeobarbus species of Lake Tana. Note that the L. intermedius SC (right-bottom) belongs to the shore complex of Lake Tana and resembles the ancestormost.

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of Deoxynucleoside Triphoshate set PCR Grade (Roche-1.969.064),0.3 ll of 5.0 U/ll Taq DNA Polymerase (Sigma-D6677) and 1� ofamplification buffer (Promega). Amplification conditions involveda first denaturation at 94 �C for 2 min, annealing at 50 �C for1 min, extension at 71 �C for 1 min, and then 30 cycles of denatur-ation at 94 �C 30 s, annealing at 50 �C for 1 min 30 s and extensionat 71 �C for 1 min (followed by a final extension at 71 �C for 3 min).For visualization, 5 ll of each PCR product was run on a 1.2% TAE-agarose gel and stained with Ethidium-Bromide. The remainingamplified product was purified using a Zymo-DNA Clean and Con-centrator/Spin I – column kit following the manufacturer’s proto-col, Zymo Research Inc. The sequencing reaction consisted ofdenaturation at 95 �C for 1 min and 25 cycles at 96 �C for 10 s, at50 �C for 5 s and at 60 �C for 4 min.

The volume of the sequencing reaction was 5 ll and consistedof 0.5 ll PCR Primer 1.6 pmol/ll, 2.5 ll 10 ng/ll DNA, 1 ll 0.5�Big Dye and 1 ll Terminator Ready Reaction Mix following themanufacturer’s protocol of ABI Prism Big Dye Terminator CycleSequencing Ready Reaction Kits of Applied Biosystems.

Products were run out on a 5% denaturing acrylamide gel (San-ver Tech) and visualized on a ABI-377 automated sequencer fol-lowing the manufacturer’s instructions. Both strands weresequenced using each one of the two initial PCR primers.

2.3. Sequence alignment and analysis

The nearly complete cytochrome b sequences (positions 61-1098) of the B. barbus cytochrome b gene, following Tsigenopoulosand Berrebi, 2000) from Ethiopian labeobarbs as well as the cyto-chrome b sequences of other African cyprinid species recovered

from GenBank/EMBL (as reported in Tsigenopoulos et al., 2002;de Graaf et al., 2007) were aligned to determine the level of se-quence divergence among various Ethiopian species and popula-tions in reference to a larger set of African cyprinid taxa.Neighbour Joining (NJ) analysis with uncorrected pairwise dis-tances was carried out using PAUP4b10 (Swofford, 2002), and aNJ bootstrap analysis was carried out with 2000 replicates. ABayesian phylogenetic analysis using MrBayes3.1 (Ronquist andHuelsenbeck, 2003) was carried out using a GTR model with agamma distribution and performing 150,000 replicates (burn inof 100,000 replicates). The Ethiopian Labeobarbus mtDNA haplo-types in addition were subjected to a median joining network anal-ysis as implemented in the program Network (Bandelt et al., 1999).

3. Results

Cytochrome b sequences were obtained for 15 large Labeobar-bus species from Lake Tana, and for L. intermedius from a numberof rivers and lakes in Ethiopia (see Table 1). Sequences correspondto GenBank Accession Nos. (GQ853201–GQ853271). The resultingtrees (Fig. 3a and b) are congruent with each other and are in com-plete agreement with Tsigenopoulos et al. (2002) with regard tothe well-supported clades. Most importantly, the Ethiopian labeo-barbs from Lake Tana, Didessa River and Lake Chamo cluster withinthe African hexaploid group as distinguished by Tsigenopouloset al. (2002). Labeobarbus intermedius specimens from Lake Chamo(Rift Lakes Drainage Basin) formed a distinct group from the largelabeobarbs from Lake Tana and Didessa River (Nile Drainage Basin).The large Labeobarbus species from the Nile Drainage Basin showed

Fig. 2. Major river systems and lakes in Ethiopia. Arrows indicate the localities at which Labeobarbus species investigated in this study were collected.

338 M. de Graaf et al. / Molecular Phylogenetics and Evolution 54 (2010) 336–343

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limited levels of genetic differentiation and Lake Tana’s Labeobar-bus were paraphyletic to the labeobarbs from Didessa River.

Seventy-one cytochrome b sequences from Ethiopian largeLabeobarbus spp. were used in the haplotype analysis, including se-quences of all 15 Labeobarbus species from Lake Tana. Out of 1038bases included, 42 were variable, and we identified 26 distinct hap-lotypes (Table 1). The distribution of haplotypes among the largeLabeobarbus is shown in Table 2. There are no haplotypes thatare diagnostic for any of the large Lake Tana Labeobarbus species.No haplotypes are shared between individuals from Lake Tanaand specimens from other lakes and rivers in Ethiopia. The LakeTana haplotypes seem therefore, based on our sample, unique.

4. Discussion

Cumulative evidence from their ecology (Sibbing and Nage-lkerke, 2001; de Graaf et al., 2008), reproductive behavior (Nage-lkerke and Sibbing, 1996; Palstra et al., 2004; de Graaf et al.,2005; Zworykin et al., 2006), morphology (Nagelkerke et al.,1994; Nagelkerke and Sibbing, 2000) and genetics (Berrebi andValiushok, 1998; Dixon et al., 1996; Kruiswijk et al., 2005) has pro-vided sufficient evidence that Lake Tana’s labeobarbs are indeed‘good species’. Reproductive isolation is well maintained by macro-and micro spatial differences in spawning areas, temporal differ-ences in spawning period and assortative mating through sizeand chemoreception (Nagelkerke and Sibbing, 1996; Palstra et al.,2004; de Graaf et al., 2003, 2005; Zworykin et al., 2006).

Genetic differentiation among the endemic large Labeobarbusspecies is low (Figs. 3 and 4). Eleven cytochrome b haplotypesare present in Lake Tana, but none is diagnostic for a particular spe-cies. Meaning that there are no monophyletic clades that are spe-cific to any of the species. A notably plausible explanation, is that

the failure to distinguish species based on cytochrome b sequencesis due to the recent origin of the species flock. Low genetic differ-entiation in mtDNA was also observed in a very young (8000 yearold) Cyprinidon species flock in the Laguna Chichancanab on theYucatan peninsula in Mexico (Strecker et al., 1996; Strecker,2006), a monophyletic cichlid species pair in Lake Ejagham, Camer-oon (Schliewen et al., 2001), a �9,000 year old Alcolapia-cichlidspecies flock (Seegers et al., 1999) and among Lake Victorian cich-lid species (Meyer et al., 1990).

The haplotypes found in Lake Tana were not found in rivers andlakes elsewhere in Ethiopia based on the sample we present in thisstudy. Conversely, none of the haplotypes found outside occurred inLake Tana. This suggests a partitioning of haplotypic variation thatcould either be explained by a single incursion of ancestral L. inter-medius in the Lake Tana drainage basin, or the disruption of an an-cient continuous riverine population by the emergence of theTissisat waterfalls. The present data does not unequivocally supporta single origin (Fig. 3a and b) of Lake Tana’s Labeobarbus speciesflock, however, the current dataset consisting of a single mitochon-drial gene is insufficient and more markers are needed to infer con-clusions about the phylogenetic relationships. Although thehaplotypes of the Lake Tana labeobarbs do not overlap with thosefrom outside the lake, the distribution of haplotypes from Lake Tanaand Didessa River suggests a common riverine origin because bothlocales are polyphyletic. There appears to be no clear-cut phyloge-ographic division as haplotypes from Lake Awassa (H21, H22 andH23) from the south of Ethiopia appear to be closely related toDidessa River and Lake Tana haplotypes, while H16 from the DidessaRiver drainage basin seems to be more closely related to some of thesouth Ethiopian (Rift Valley) haplotypes. By contrast, Lake Tana’shaplotypes seem to form a subset of the total variation that occursin the Nile drainage basin, adding evidence that the Lake Tana spe-cies flock originates from a limited stock of riverine L. intermedius.

Similar to the East African haplochromine monophyletic super-flock (Nagl et al., 2000; Verheyen et al., 2003), among the barbsthere is a striking discrepancy between molecular and morpholog-ical variation. On the one hand in Lake Tana the labeobarbs di-verged morphologically to the degree of clearly distinct species(Nagelkerke et al., 1994; Nagelkerke and Sibbing, 2000). At themolecular level, by contrast, the members of the flock display con-siderable trans-specifically shared haplotypes but no fixed substi-tutions differentiating the distinct morphological forms.Determining the age of the species flock by a molecular clock anal-ysis is a trivial pursuit because of the likelihood that shared ances-tral polymorphies remain in the populations studied. At best, wecan determine the maximum age of the haplotypic variation inthe lake. With a maximum sequence divergence of approximately1% between Lake Tana haploytpes (Fig 3) and an estimated se-quence divergence of 1.3–3.3% per million years (Perdices et al.,2002 and references therein; Burridge and White, 2000 and refer-ences therein) the Lake Tana species flock can be estimated to benot older than 770,000–300,000 years. This contrasts with Dixonet al. (1996) and Kruiswijk et al. (2005), who found that lineagesin the MHC class 2b could be up to 5 Ma old or even older.

Other studies have shown that the age of divergence of mito-chondrial lineages can be a poor estimate for the age of a speciesflock in recently formed (refilled) lakes. In Lake Victoria haplochro-mine cichlids, for instance, lineage diversification at 100,000 yearsago (Nagl et al., 2000; Verheyen et al., 2003) is clearly in excess ofthe age of the (current) lake, estimated at approximately15,000 years old (Johnson et al., 1996, 2000). Given the poor phy-logenetic structure between Didessa River and Lake Tana haplo-types, it seems plausible that the haplotypic variation of LakeTana did not originate after the lake emerged. The upper boundof 770,000 years BP, therefore, is likely to be an overestimation ofthe age of (the species flock in) Lake Tana.

Table 1Haplotypes found in the endemic large Labeobarbus spp from Lake Tana and L.intermedius collected in several river and lakes in Ethiopia. Position numbers refer tothe B. barbus cytochrome b sequence (AF112123; Tsigenopoulos and Berrebi, 2000).

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0.01 substitutions/site

Cyclocheilichthys sp. (ASIA)

Barbus mattozi (MOZAMBIQUE)

Barbus pleurogramma (L. TANA)

Moxostoma breviceps (N-AMERICA)

African large hexaploids

African sm

all diploidsS. A

frican tetraploids

African sm

all diploids

non-Cyprinines

Catostomidae

Labeobarbus sp. (L. TANA)Labeobarbus acutirostris (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus platydorsus (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus intermedius SC (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus nedgia (L. TANA)Labeobarbus brevicephalus (L. TANA)Labeobarbus platydorsus (L. TANA)Labeobarbus acutirostris (L. TANA)

Labeobarbus intermedius (DIDESSA R.)Labeobarbus intermedius (DIDESSA R.)

Labeobarbus gorgorensis (L. TANA)Labeobarbus nedgia (L. TANA)Labeobarbus latydorsus (L. TANA)Labeobarbus brevicephalus (L. TANA)Labeobarbus nedgia (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus crassibarbis (L. TANA)Labeobarbus gorgorensis (L. TANA)Labeobarbus crassibarbis (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus gorguari (L. TANA)

Labeobarbus intermedius (L. Chamo)Labeobarbus intermedius (L. Chamo)Labeobarbus intermedius (L. Chamo)Labeobarbus intermedius (L. Chamo)

Labeobarbus bynni occidentalis

Labeobarbus marequensis (S-AFRICA)Labeobarbus ethiopicus (S-AFRICA)

Labeobarbus polylepis (S-AFRICA)Labeobarbus capensis (S-AFRICA)

Barbus reinii (MOROCCO)Barbus luteus (ASIA-MINOR)

Barbus barbus (EUROPE)Barbus barbus (EUROPE)

Barbus cyclolepis cyclolepis (EUROPE)Barbus meridionalis (EUROPE)

Barbus bocagei (EUROPE)Aulopyge huegelii (EUROPE)Cyprinion macrostomus (ASIA-MINOR)

Schizothorax pseudoaksaiensis (ASIA)Schizothorax prenanti (ASIA)

Puntius schwanenfeldii (ASIA)Carassius auratus

Carassius auratus langsdorfiCyprinus carpio

Puntius gonionotus (ASIA)

Barbus ablabes (IVORY COAST)Barbus macrops (GUINEA)

Barbus guineensis (GUINEA)Barbus cadenati (SIERRA LEONE)

Barbus kerstenii (KENYA)Barbus trimaculatus (S-AFRICA)

Barbus humilis (L. TANA) Barbus humilis (L. TANA)Barbus tanapelagius (L. TANA)Barbus humilis (L. TANA)Barbus tanapelagius (L. TANA)Barbus tanapelagius (L. TANA) Barbus tanapelagius (L. TANA) Barbus humilis (L. TANA)

Barbus nyanzae (KENYA)Barbus sublineatus (IVORY COAST)

Pseudobarbus burchelli (S-AFRICA)Pseudobarbus burgi (S-AFRICA)

Pseudobarbus asper (S-AFRICA)Pseudobarbus afer (S-AFRICA)

Barbus trevelyani (S-AFRICA)Barbus andrewi (S-AFRICA)Barbus serra (S-AFRICA)

Barbus erubescens (S-AFRICA)Barbus calidus (S-AFRICA)

Barbus anoplus (S-AFRICA)Barbus paludinosus (L. Awassa)Barbus paludinosus (L. Ziway)Barbus paludinosus (L. Awassa)Barbus paludinosus (L. Ziway)

Barbus paludinosus (L. Awassa)Barbus paludinosus (L. Ziway)Barbus paludinosus (L. Ziway)

Barbus paludinosus (L. Awassa)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (DIDESSA R.)

Barbus paludinosus (DIDESSA R.)Barbus pleurogramma (L. TANA)Barbus pleurogramma (L. TANA)Barbus pleurogramma (L. TANA)

Garra variabilis (ASIA-MINOR)Leuciscus leuciscus (EUROPE)

Gobio gobio (EUROPE)Tinca tinca (EUROPE)

Myxocyprinus asiaticus (ASIA)

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Fig. 3. (a) Neighbour Joining tree of the Ethiopian Labeobarbus species, Ethiopian Barbus species (taken from de Graaf et al., 2007) and other predominantly African cyprinidfishes (taken from Tsigenopoulos et al., 2002) based on cytochrome b sequence data. Branch lengths are proportional to the mean number of substitutes per site (see scalebar). Numbers above nodes indicate bootstrap values higher than 50%. Sequences of two catostomid taxa were used as an outgroup. (b) Bayesian phylogenetic analysis of thesame cyprinid cytochrome b sequence data. Numbers above the node indicate Bayesian posterior probabilities.

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Adaptive radiation and speciation within Lake Tana’s Labeobar-bus species flock most likely occurred in the last 10,000–25,000 years based on the low sequence differentiation in mtDNAand corroborating geological evidence. East Africa is prone to largedrastic changes in climate and rainfall, resulting in frequent fluctu-ations in water levels in the lakes in this region (Owen et al., 1990).In Lake Tanganyika (max depth 1400 m, average depth 570 m)

water levels were 600 m below the current level approximately25,000 years ago (Danley and Kocher, 2001), while Lake Victoria(max depth 70 m, average depth 40 m) might have dried out com-pletely around 15,000 years ago (Johnson et al., 1996, 2000). Com-pared to these other East African lakes, the saucer-shaped LakeTana is very shallow (max depth 14 m, average depth 8 m) and itis therefore no surprise that Lamb et al. (2007) found evidence

Labeobarbus intermedius SC (L. TANA)Labeobarbus nedgia (L. TANA)Labeobarbus platydorsus (L. TANA)Labeobarbus brevicephalus (L.TANA)Labeobarbus intermedius (DIDESSA RIVER)Labeobarbus intermedius (DIDESSA RIVER)Labeobarbus nedgia (L. TANA)Labeobarbus brevicephalus (L. TANA)Labeobarbus nedgia (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus gorguari (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus macrophtalmus (L. TANA)Labeobarbus crassibarbis (L. TANA)Labeobarbus gorgorensis (L. TANA)Labeobarbus crassibarbis (L. TANA)Labeobarbus acutirostris (L. TANA)Labeobarbus acutirostris (L. TANA)Labeobarbus tsanensis (L. TANA)Labeobarbus platydorsus (L. TANA)Labeobarbus platydorsus (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus megastoma (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus truttiformis (L. TANA)Labeobarbus intermedius SC (L. TANA)Labeobarbus gorgorensis (L. TANA)Labeobarbus intermedius (L. CHAMO)Labeobarbus intermedius (L. CHAMO)Labeobarbus intermedius (L. CHAMO)Labeobarbus intermedius (L. CHAMO)Labeobarbus bynni occidentalisLabeobarbus ethiopicus (ETHIOPIA)Labeobarbus marequensis (S-AFRICA)Labeobarbus polylepis (S-AFRICA)Labeobarbus capensis (S-AFRICA)Barbus reinii (MOROCCO)Barbus luteus (ASIA-MINOR)Puntius schwanenfeldii (ASIA)Barbus ablabes (IVORY COAST)Barbus macrops (GUINEA)Barbus cadenati (SIERRA LEONE)Barbus guineensis (GUINEA)Barbus mattozi (MOZAMBIQUE)Barbus trimaculatus (S-AFRICA)Barbus humilis (L. TANA)Barbus humilis (L. TANA)Barbus humilis (L. TANA)Barbus tanapelagius (L. TANA)Barbus tanapelagius (L. TANA)Barbus tanapelagius (L. TANA)Barbus tanapelagius (L. TANA)Barbus humilis (L. TANA)Barbus nyanzae (KENYA)Barbus sublineatus (IVORY COAST)Barbus anoplus (S-AFRICA)Barbus kerstenii (KENYA)Barbus paludinosus (L. AWASSA)Barbus paludinosus (L. ZIWAY)Barbus paludinosus (L. AWASSA)Barbus paludinosus (L. AWASSA)Barbus paludinosus (L. ZIWAY)Barbus paludinosus (L. ZIWAY)Barbus paludinosus (L. ZIWAY)Barbus paludinosus (L. AWASSA)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (DIDESSA R.)Barbus paludinosus (GIBE R.)Barbus pleurogramma (L. TANA)Barbus pleurogramma (L. TANA)Barbus pleurogramma (L. TANA)Barbus pleurogramma (L. TANA)Pseudobarbus burchelli (S-AFRICA)Pseudobarbus burgi (S-AFRICA)Pseudobarbus asper (S-AFRICA)Pseudobarbus afer (S-AFRICA)Barbus andrewi (S-AFRICA)Barbus serra (S-AFRICA)Barbus erubescens (S-AFRICA)Barbus calidus (S-AFRICA)Barbus trevelyani (S-AFRICA)Barbus barbus (EUROPE)Barbus barbus (EUROPE)Barbus cyclolepis cyclolepis (EUROPE)Barbus meridionalis (EUROPE)Barbus bocagei (EUROPE)Aulopyge huegelii (EUROPE)Cyprinion macrostomus (ASIA-MINOR)Schizothorax pseudoaksaiensis (ASIA)Schizothorax prenanti (ASIA)Puntius gonionotus (ASIA)Carassius auratusCarassius auratus langsdorfiCyprinus carpioCyclocheilichthys sp (ASIA)Garra variabilis (ASIA-MINOR)Leuciscus leuciscus (EUROPE)Tinca tinca (EUROPE)Gobio gobio (EUROPE)Myxocyprinus asiaticus (ASIA)Moxostoma breviceps (N-AMERICA)

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100100

10097

100

100

100

100

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5857

100

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85

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10082

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9997

99 100

82

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10072 84

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10094

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89100

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96 100

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57 88 100

100 93

Catostomidae

Non-Cyprinines

S-Africantetraploids

African “small”diploids

African “largel”hexaploids

Fig. 3 (continued)

M. de Graaf et al. / Molecular Phylogenetics and Evolution 54 (2010) 336–343 341

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for the desiccation of Lake Tana around 17,000 years ago, at thesame time as Lake Victoria.

Furthermore, the lake might owe its present form to dammingby a 50-km-long Quaternary basalt flow, which filled the exit chan-nel of the Blue Nile river to a possible depth of 100 m (Grabhamand Black, 1925; Jespen and Athearn, 1961). The age of this lavaflow is estimated to be some 10,000 B.P. (Grabham and Black,1925). However, according to Chorowicz et al. (1998) the geomor-phology of Lake Tana is not consonant with a lava-dammed,flooded river valley. In his view the present-day morphology ex-presses a central focus of subsidence, despite fault reactivationand headward downcutting of the Blue Nile. Whether or not the

lake was present before or after the blocking of the Nile outlet,the lava flow did create the 40-m high waterfalls at Tissisat, isolat-ing Lake Tana from the lower Nile drainage.

Current research focuses on additional geographic sampling tostrengthen our hypothesis that Lake Tana’s Labeobarbus speciesflock is very young (�15,000 years old), of monophyletic originand derived from the Nile basin. Furthermore resolving the phylog-eny of Lake Tana’s Labeobarbus species flock using more sensitivemolecular techniques would allow for testing our hypothesizedevolutionary scenario, i.e. sequential episodes of diversification(habitat followed by trophic morphology) and the replicated evolu-tion of trophic specializations in different habitats (de Graaf et al.,

Table 2Frequency distribution of the 26 identified (see Table 1) haplotypes among the large Labeobarbus spp. collected the different lake and rivers in Ethiopia. No = number ofspecimens.

Species Location No Haplotypes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

L. acutirostris Lake Tana 3 1 2L. brevicephalus Lake Tana 2 1 1L. crassibarbis Lake Tana 3 2 1L. dainellii Lake Tana 2 1 1L. gorgorensis Lake Tana 3 1 1 1L. gorguari Lake Tana 2 2L. intermedius SC Lake Tana 3 2 1L. longissimus Lake Tana 1 1L. macrophtalmus Lake Tana 3 2 1L. megastoma Lake Tana 3 2 1L. nedgia Lake Tana 3 1 1 1L. platydorsus Lake Tana 3 1 1 1L. surkis Lake Tana 1 1L. truttiformis Lake Tana 3 2 1L. tsanensis Lake Tana 3 1 1 1L. intermedius Didessa River 18 7 6 1 1 1 1 1L. intermedius Didessa River (Lugo trib.) 4 1 1 1 1L. intermedius Didessa River (Dabena trib.) 1 1L. intermedius Lake Awassa 4 2 1 1L. intermedius Bulbula River 1 1L. intermedius Lake Chamo 4 4L. intermedius Arer River 1 1

71 10 8 7 3 3 2 1 1 1 1 1 8 7 1 1 2 1 1 1 1 2 1 1 1 1 4

H13

H12

H2

H3

H1

H6

H16

H4

H5

H26

H25

H24 H22

H23

H20H15

H18

H14H19

H8

H7

H11

H10

H21

H9Lake TanaDidessa RiverLake AwassaBulbula River

Arer RiverLake Chamo

Fig. 4. Unrooted haplotype network for the Labeobarbus species flock and other Ethiopian Labeobarbus species. Each line in the network represents a single mutational step.Size of the oval represents the number of individuals (minimum 1–maximum 10) sharing that haplotype. Black circles represent haplotypes that were not found in thesample.

342 M. de Graaf et al. / Molecular Phylogenetics and Evolution 54 (2010) 336–343

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2008). Resolving the phylogenetic relationships of Lake Tana’sLabeobarbus species using other (especially nuclear) markers couldprovide a better understanding of the evolutionary patterns andmechanisms in niche segregation, driving morphological diversifi-cation and speciation.

Acknowledgments

We would like to thank the Amhara Regional Agricultural Re-search Institute in Bahir Dar (Ethiopia) for facilitating the Labeoar-bus project. Furthermore we would like to thank Trudi Hermsenfor her assistance with sequencing and Erika Nentwich for her sup-port in Ethiopia and in the Netherlands. The research was fundedby the Netherlands Foundation for the Advancement of TropicalResearch, NWO-WOTRO project W 82–231 (de Graaf).

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