Historical Biogeography of Fishes Of

ABSTRACT

The purpose of this study is to elucidate the historical processes that have impacted the fishes of the Fouta Djallon highlands and surrounding areas. This mountainous region in

Guinea, West Africa, lies on the northern edge of the Guinean Range. This geologic formation, of Jurassic origins, has long reported to serve as a barrier to dispersal in the region. These highlands currently separate two ichthyo-provinces in the area. The upper-

Guinean province encompasses rivers on the Western slopes of the range that flow directly into the Atlantic Ocean. The Nilo-Sudan province is comprised of the rivers and streams on the Eastern slopes of the Guinean Range. These rivers flow west or north through the Sahel and eventually back to the Atlantic Ocean. While these highlands clearly serve as a barrier to dispersal for most fish taxa, some taxa are reported to occur on both sides of the Fouta Djallon. This study investigates three groups of these “amphi-

Guinean” taxa to determine if the same taxa are present within both provinces and what processes would have allowed for this dispersal to take place. In addition to the biogeographical questions addressed within the Fouta Djallon region, specimens from the surrounding areas are included to further understand the historical biogeography of these groups in West Africa. This study revealed the presence of numerous undescribed species within the Amphilius, Chiloglanis, and ‘Barbus’ groups investigated. While some taxa do appear to be amphi-Guinean others are restricted to one ichthyo-province or the other.

Numerous headwater capture events within the area have allowed taxa to expand ranges and diversify. This study also provides insights on areas of endemism within the region where additional undiscovered diversity is likely to occur.

ACKNOWLEDGEMENTS

I would like to thank my wonderful fiancée, Michelle, and my family for all of their support over the years. I would also like to thank my advisor, Henry L. Bart Jr, for all of the opportunities provided and the assistance given. Committee members Michael

J. Blum, Steven P. Darwin, and Frank Pezold, provided many wonderful discussions and comments that greatly improved this study.

Support during my tenure at Tulane was provided through teaching assistantships through the department of Ecology and Evolutionary Biology and research assistantships.

These NSF funded assistantships (MCB# 1027830, OISE: 0968727) greatly improved my research over the years. I would also like to thank the All Cypriniformes Species

Inventory (ACSII, DEB #1023403) for funding the 2013 expedition to Guinea, West

Africa. This work would not have been possible without this support.

I have been fortunate enough to work with many great people of the years at

Tulane. I want to thank the following people for all their assistance in the lab and in the field: Larcie, Burnett, Mike Doosey, Justin Mann, DJ Abibou, Nelson Rios, Paulette

Reneau, David Lach, John P. Friel, Jon W. Armbruster, Malorie Hayes and all of the undergraduate students that have assisted in the lab over the years. I would also like to thank my good friends here in New Orleans, back in Corpus Christi, and wherever you have moved on too. My first visit to Africa was largely at the behest of Zeno Wicks,

South Dakota State University, and for this I am forever grateful.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... v

LIST OF FIGURES...... vi

CHAPTER 1. Introduction...... 1 Hydrological History ...... 3 Study taxa...... 5 Significance of proposed research ...... 9 Hypotheses and tests ...... 12

CHAPTER 2. Phylogeography of the suckermouth catfishes (Mochokidae: Chiloglanis) from the Fouta Djallon highlands and surrounding areas ...... 24 Abstract ...... 24 Introduction ...... 24 Materials and Methods ...... 26 Results ...... 29 Discussion ...... 32 Conclusions ...... 37

CHAPTER 3. Phylogeography and diversity of mountain catfishes (Amphiliidae: Amphilius) from Guinea, West Africa ...... 57 Abstract...... 57 Introduction ...... 57 Materials and Methods ...... 60 Results ...... 62 Discussion ...... 68 Conclusions ...... 74

CHAPTER 4. Phylogeography of certain small barbs (Cyprinidae: ‘Barbus’) from the Fouta Djallon highlands in Guinea, West Africa ...... 99 Abstract ...... 99 Introduction ...... 99 Materials and Methods ...... 101 Results ...... 104 Discussion ...... 107 Conclusions ...... 110

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CHAPTER 5. Comparative phylogeography and historical biogeography of the fishes of the Fouta Djallon and surrounding areas ...... 127 Abstract ...... 127 Introduction ...... 127 Methods...... 129 Results ...... 131 Discussion ...... 131

REFERENCES ...... 142

LIST OF TABLES

Table 2.1. Chiloglanis specimens included in the molecular analysis (species name, collection locality, voucher No or field No, GenBank accession No for Cyt b, GenBank accession No for GH intron 4. Specimens will be associated with field numbers when deposited at genetic repositories ...... 39 Table 3.1. Amphilius specimens included in the molecular analysis (species name, collection locality, drainage, voucher No or field No, GenBank accession No for Cyt b, GenBank accession No for GH intron 4. Specimens will be associated with field numbers when deposited at genetic repositories ...... 76

Table 3.2. Major events of divergence within the A. grammatophorus and A. platychir groups...... 80 Table 4.1. ‘Barbus’ specimens included in the molecular analysis (species name, collection locality, source (A - Macordom & Doadrio 2001a, B - Macordom & Doadrio 2001b, C- Tsigenopoulos et al. 2002) voucher No or field No, GenBank accession No for Cyt b. Specimens will be associated with field numbers when deposited at genetic repositories...... 112

Table 5.1. Table 5.1 Venn diagram for each taxa coded for area ...... 135

LIST OF FIGURES

Figure 1.1. The rivers of Africa with demarcation between High and Low Africa. The study area, Guinea, outlined in black ...... 18

Figure 1.2. Three major river’s headwaters arise in Guinea, West Africa. The Niger River flows northeast and then turns southwest towards the Atlantic. The Senegal and Gambie rivers flow northwest into the Atlantic Ocean ...... 19

Figure 1.3. Rivers of the Fouta Djallon highlands and surrounding areas. Generalized extent of Fouta Djallon highlands denoted by dotted-line. Extent of the three proposed ichthyological provinces (Upper Guinean, Nilo-Sudan, and Eburneo- Ghanean) within dashed line marking the boundaries ...... 20

Figure 1.4. Amphilius cf. platychir from Fouta Djallon (Badi River, bar = 1 cm). Note the dorsal- ventrally compressed profile and broad stiffened pectoral fins ...... 21

Figure 1.5. Chiloglanis cf. occidentalis from the Fouta Djallon (Senegal River basin, bar = 1 cm). Note the distinctive oral disc, dorsal-ventrally compressed body, and spines in dorsal and pectoral fins ...... 22

Figure 1.6. Representative ‘Barbus’ spp. from the Fouta Djallon which display a variety of pigmentation patterns, meristic counts, and external morphological features that aid in identifying individual species. A) ‘Barbus’ cadenati from the Gambie River, B) ‘B.’ guineensis from the Kokoulo River (note the serrations on third ray in dorsal fin), C) male ‘B.’ pobeguini from the Kokoulo, D) female ‘B.’ pobeguini from the Kokoulo River (note the absence of barbels and the incomplete lateral line, bar = 1cm) ...... 23 Figure 2.1. Rivers of the Fouta Djallon highlands and surrounding areas. Generalized extent of Fouta Djallon highlands denoted by dotted-line. Extent of the three proposed ichthyological provinces (Upper Guinean, Nilo-Sudan, and Eburneo- Ghanean) within dashed line marking the boundaries ...... 42

Figure 2.2 Localities (open circles) where Chiloglanis specimens (filled circle) and tissue samples (star) were collected during the 2003 and 2013 expeditions. Extent of proposed ichthyological provinces denoted by dashed lines and water falls indicated by hatched lines ...... 44

Figure 2.3. Phylogeny of Chiloglanis spp. inferred from cytochrome b with posterior probabilities above and bootstrap support below (>95 and >.95 represented by *). Gray branches collapse (bs <50) in maximum likelihood analysis and average uncorrected distance between clades shown at node...... 45

Figure 2.4. Phylogeny of Chiloglanis spp. inferred from concatenated (Cyt b, GH intron 4, and character derived from indels) dataset with posterior probabilities above and bootstrap support (bs) below (> 95 and .95 represented by *). Gray branches (bs <50) collapse in maximum likelihood analysis...... 46

Figure 2.5 Chronogram of Chiloglanis spp. inferred from BEAST analysis of concatenated dataset (Cyt b and GH intron 4). Estimated age (millions of years) is given and gray bars represent 95% confidence intervals (HPD: Highest Posterior Distributions) ...... 47

Figure 2.6. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). A) Chiloglanis sp. “Konkouré” and B) Chiloglanis sp. “Badi ...... 48 Figure 2.6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). C) Chiloglanis sp. “Little Scarcies” and D) Chiloglanis sp. “Kolenté”...... 49 Figure 2.6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). E) Chiloglanis sp. “Moa R.” and F) Chiloglanis sp. “Senegal/Niger ...... 50 Figure 2.6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). G) Chiloglanis sp. “Loffa” and H) Chiloglanis sp. “St. Paul ...... 51 Figure 2.6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). I) Chiloglanis sp. “St. John” and J) Chiloglanis sp. “Loffa sp. 2” ...... 52

Figure 2. 6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm). K) Chiloglanis lamottei and L) Chiloglanis cf. micropogon ...... 53 Figure 2.6 cont. Lateral and ventral images of representative morphotypes from each Chiloglanis species identified in this study (scale bar = 1 cm): M) Chiloglanis sp. “Moa R. long spine” ...... 54

Figure 2.7. Distribution patterns of Chiloglanis lamottei, Chiloglanis cf. micropogon, and the “short spine” Chiloglanis species discovered in this study. Dashed line denoted boundaries of proposed ichthyo-provinces ...... 55

Figure 2.7. Distribution patterns of the “long spine” Chiloglanis species discovered in this study. Dashed line denoted boundaries of proposed ichthyo-provinces ...... 56

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Figure 3.1. Localities (open circles) where Amphilius specimens (filled circle) and tissue samples (star) were collected during the 2003 and 2013 expeditions. Extent of proposed ichthyological provinces (upper Guinean, Nilo-Sudan, Eburneo-Ghanean) denoted by dashed lines...... 82 Figure 3.2. Rivers of the Fouta Djallon highlands. Hatched lines indicate waterfalls and broken line denotes boundary between the upper Guinean ichthyo-province to the west and the Nilo-Sudan province on the east...... 83

Figure 3.3. Phylogeny inferred from maximum likelihood and Bayesian inference of cytochrome b data from all Guinean Amphilius and representatives from East and Central Africa. Branch support (poster probability/bootstrap support) listed above the branches of main clades identified (*>.95/95)...... 84 Figure 3.4. Phylogeny of the Amphilius grammatophorus and Amphilius platychir groups inferred from ML and BI analysis of partial cytochrome b. Branch support (posterior probabilities/bootstrap support) listed on branches (* indicates support > .95/95) and uncorrected p-distance between groups listed at nodes (A). Basal topology recovered from ML analysis (B), note how Amphilius sp. from the Loffa river is sister to the Amphilius platychir group in the ML analysis ...... 85

Figure 3.5. Phylogeny of Amphilius rheophilus and A. atesuensis groups inferred from ML and BI analysis of partial Cyt b. Branch support (posterior probabilities/bootstrap support) listed on branches (* indicates support > .95/95) and uncorrected p-distance between groups listed at nodes...... 87

Figure 3.6 Phylogeny of Amphilius spp. inferred from concatenated dataset (Cyt b, GH intron 4, and characters derived from gap coding ambiguous areas (indels) within intron 4), branch support (posterior probability/bootstrap support) listed on branches (* indicates support > .95/95) ...... 88

Figure 3.7. Chronogram of Amphilius grammatophorus and A. platychir groups inferred from BEAST analysis of Cyt b. Estimated age (Millions of years) is given and gray bars represent 95% confidence intervals (HPD: Highest Posterior Distributions ...... 89 Figure 3.8. Chronogram of the Amphilius rheophilus and A. atesuensis group inferred from BEAST analysis of cytochrome b. Estimated age (Millions of years) is given and gray bars represent 95% confidence intervals (HPD: Highest Posterior Distributions ...... 90

Figure 3.9. Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). A) Amphilius cf. grammatophorus “St John”, B) Amphilius sp. “Loffa”, C) Amphilius sp. “Badi/Fatala”, and D) Amphilius cf. platychir “Kolenté” ...... 91

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Figure 3.9 cont. Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). E) Amphilius cf. grammatophorus “Badi”, F) Amphilius cf. grammatophorus “Mayonkouré sp. 1”, G) Amphilius cf. grammatophorus “Mayonkouré sp. 2”, and H) Amphilius grammatophorus s.s. from Kokoulo River (type locality) ...... 92

Figure 3.9 cont. . Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). I) Amphilius cf. grammatophorus “Sala”, J) Amphilius cf. grammatophorus “Konkoure”, K) Amphilius kakrimensis from type locality and L) Amphilius cf. grammatophorus “Senegal” ...... 93

Figure 3.10. Distribution of species discovered in this study within the previously recognized taxa, Amphilius grammatophorus and A. platychir ...... 94 Figure 3.11. Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). A) Amphilius cf. rheophilus “St John”, B) Amphilius rheophilus s.s. from the Rio Corubal, C) Amphilius cf. rheophilus “Konkouré”, and D) Amphilius cf. rheophilus “Kokoulo” ...... 95 Figure 3.11 cont. Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). E) Amphilius cf. rheophilus “Senegal”, F) Amphilius cf. rheophilus “Little Scarcies”, G) Amphilius cf. rheophilus “Niger”, and H) Amphilius cf. rheophilus “Moa River”...... 96 Figure 3.11 cont. Lateral images of representative morphotypes from Amphilius spp. identified in this study (scale bar = 1 cm). I) Amphilius cf. rheophilus “Loffa” and J) Amphilius cf. rheophilus “St. Paul” ...... 97

Figure 3.12. Distribution of species discovered in this study within the previously recognized Amphilius rheophilus...... 98 Figure 4.1. Localities within Guinea were the serrated barbs were collected and tissues taken. Hatching within a river indicates a water fall...... 116

Figure 4.2 Localities where the reduced barbs were collected and tissue samples retained. Hatching within river indicates a waterfall...... 117 Figure 4.3. Saturation plots for each codon position. Black (x) indicates transitions while gray triangles indicate tranversions...... 118

Figure 4.4. Phylogeny of ‘Barbus’ inferred from cytochrome b, bootstrap support (1000 bs) above branches. Note both the reduced barb and serrated barb group are recovered as monophyletic ...... 119

Figure 4.5. Phylogeny inferred from maximum likelihood and Bayesian inference of cytochrome b data from the “serrated barb” (A) and “reduced barb” (B) groups. Branch support (poster probability/bootstrap support) listed above the branches (* indicates support >.95/95) and uncorrected p-distance between clades shown at nodes...... 120

Figure 4.6. Phylogeny of ‘Barbus’ spp. inferred from concatenated dataset (Cyt b, GH intron 2, and characters derived from gap coding ambiguous areas (indels) within intron 2), branch support (bootstrap support) listed on branches. Note both the reduced barb and serrated barb groups are recovered as monophyletic ...... 121 Figure 4.7. Chronogram of the serrated barbs (A) and the reduced barbs (B) inferred from the concatenated dataset. Estimated age (in Millions of years) is given and gray bars represent 95% confidence intervals (HPD: Highest Posterior Distributions)...... 122

Figure 4.8. Morphotypes of the serrated ‘Barbus’ discovered in this study (bar = 1 cm). A) ‘Barbus’ cadenati from the Gambie River, B) ‘B.’ dialonensis from the Senegal River, C) ‘B.’ guineensis from the Kokoulo River, and D) ‘B.’ guineensis from the Sala (Kakrima) River...... 123 Figure 4.9. Morphotypes of the reduced ‘Barbus’ taxa discovered in this study. A) ‘B.’ leonensis, B) ‘B.’ pobeguini from the Rio Corubal, C) male ‘B.’ cf. salessei from the Kakrima River, and D) male ‘B’ salessei from the Kokoulo River (note the absence of barbels and the incomplete lateral line, bar = 1cm)...... 124 Figure 4.10. Reconstruction of the biogeographical history of the serrated barb taxa (A) with current distributions (B)...... 125 Figure 4.11. Distributions of reduced barb (‘Barbus’) taxa discovered in this study...... 126 Figure 5.1. The Fouta Djallon highlands and surround areas, note the presence of the Guinean Range as it stretches from the Fouta Djallon region to Mt. Nimba ...... 136 Figure 5.2. Area cladograms for each of the study taxa, A) Amphilius grammatophorus, B) Chiloglanis, C) A. rheophilus, D) A. atesuensis, E) serrated ‘Barbus’ group, and F) reduced ‘Barbus’ group...... 137 Figure 5.2 cont. Area cladograms for each of the study taxa, D) A. atesuensis, E) serrated ‘Barbus’ group, and F) reduced ‘Barbus’ group...... 138 Figure 5.3. Combined chronogram for all study taxa to identify areas of pseudo- congruence...... 139 Figure 5.4. General Area Cladogram for co-distributed taxa constructed through the PACT algorithm...... 140

CHAPTER 1

INTRODUCTION

An estimated 3000 species of freshwater fishes inhabit the lakes and rivers of

Africa (Lévêque 1997). Although much interest is devoted to the diversity observed in the Great Lakes of the African Rift system, the majority of fish species occur in the rivers and streams of sub-Saharan Africa (Figure 1.1). The rate of species discovery in recent years suggests that many taxa remain to be discovered and formally described (Skelton &

Swartz 2011). Africa can be divided into two general areas; high Africa, to the east and south, comprises the great lakes and rivers and streams above 1000m. Low Africa, to the north and west, mainly comprises sedimentary basins and upland plains generally below

600m (Beadle 1971, Figure 1.1). The diversity within the rivers of high Africa is much lower than the diversity within low Africa (Skelton and Swartz 2011). Ongoing research in low Africa continues to result in the discovery of new taxa (Bamba et al. 2011; Decru et al. 2011; 2013).

The Fouta Djallon highlands in Guinea, West Africa – regarded as part of Low

Africa - rise over 1000 m and serve as the headwater source to several major rivers in

West Africa, including the Gambie, Senegal, and Tinkisso (an upper tributary to the

Niger River, Figure 1.2).). This area is also the source of the Rio Corubal, Konkouré,

Little Scarcies, and other smaller rivers that flow westward into the Atlantic Ocean

(Figure 1.3). The importance of these rivers to the surrounding countries earns Guinea the

2 nickname, Château d'eau de l'Afrique de l'ouest (the water tower of West Africa). Along with the rivers originating in the Fouta Djallon there are also many rivers that occur in southeastern Guinea (région Forestière, Figure 1.3). Once lined with gallery forests or found within rain forests, the streams and rivers of this region are facing numerous threats (Thieme et al. 2005). To the northwest of Mt. Nimba are streams that either flow to the Atlantic Ocean (through Liberia or Sierra Leone) or northward to the Niger River.

A high number of endemics inhabit this region (Daget 1962; Teugels et al. 1987;

Howes and Teugels 1989; Romond 1994; Vreven and Teugels 2005). Many of these species inhabit streams on the western slopes of the Fouta Djallon that drain into the

Atlantic Ocean. Another area of high endemism is the Mt. Nimba area. The number of aquatic endemic species around Mt. Nimba is quite surprising, including an estimated 81 odonate species, an aquatic shrew, viviparous frogs, and several crustacean species

(Thieme et al. 2005). The high number of endemic species within the Fouta Djallon, the region’s Eocene age (Michel 1973) and its relative environmental stability, have prompted its designation as a refugium (Mayr and O’Hara 1986).

The biogeography of the Fouta Djallon highlands, like that of most of Africa, is not well understood. African ichthyological provinces have been proposed numerous times based upon faunal similarities across various drainage basins (Boulenger 1905;

Nichols and Griscom 1917; Nichols 1928; Worthington 1954; Roberts 1975). Subsequent workers have clarified the delineations between some of these provinces, but lacked species phylogenies to generate hypotheses of basin relationships (Hugueny 1990;

Hugueny and Lévêque 1994). The Nilo-Sudan and Upper Guinean ichthyo-provinces meet in the Fouta Djallon (Roberts 1975), with different species occurring on either side

3 of the boundary. However, other species occur are known to occur on both sides of the divide. A biogeographic hypothesis that accounts for these so-called amphi-Guinean

(Daget 1962) species has not yet been proposed.

A more complicated history of the area has emerged as phylogenies and morphological analyses of certain taxa have been produced. For example, Amphilius rheophilus populations from the western slope of the Fouta Djallon (Konkouré River) are divergent from those on the northern slope (Gambie, Rio Corubal), populations from the eastern slope (Senegal and Niger Rivers), and populations on the southern slope (Little

Scarcies River, Schmidt and Pezold 2011, Figure 1.3). In addition to the diversity revealed within described Amphilius spp. from the area; many new endemic species have been described since Daget’s (1962) seminal work on the area (Teugels et al. 1987;

Howes and Teugels 1989; Romond 1994; Vreven and Teugels 2005; Bamba et al. 2011).

Based on these discoveries, it is quite likely that additional fish diversity awaits discovery within the Fouta Djallon and surrounding areas.

Hydrological history

The Fouta Djallon highlands first rose by epeirogenic uplift of the Guinean Range in the late Jurassic (160 – 104 Ma) and were further accentuated by subsidence of the western portion of the region (Michel 1973). The Guinean range stretches from the Fouta

Djallon southeast to Mt Nimba (Figure 1.3). This uplift caused the Gambie and the

Bafing rivers to flow in a northeastward direction and the Konkouré, Kakrima and

Koumba rivers to flow west or southwest. The Bafing River at this time probably flowed into a large endorheic basin just northeast of the Fouta Djallon (Michel 1973). A second

4 uplift during the upper Eocene (39 – 33 Ma) caused the Bakoy and Bafing Rivers to join and form the Senegal, which flowed northwest towards the sea (Michel 1973).

The topography of the Fouta Djallon remained relatively staid until the second half of the upper Pleistocene (Michel 1973). During a humid phase (40,000 BP); rivers became deeply incised throughout their courses, but most notably in their headwaters

(Lévêque 1997). During the last dry phase (14,000-12,000 BP) large dunes pushed southward and blocked off the Senegal River, causing it to become endorheic. It is possible that both the Gambie and Senegal rivers also became endorheic during the last interpluvial phase. The subsequent pluvial phase could have allowed faunal exchanges between lower courses of the Gambie and Senegal rivers (Roberts 1975).

The headwaters of the Niger River, West Africa’s longest river, originate near the

Fouta Djallon, only 250 km from the Atlantic Ocean. From this point, the Niger currently flows northeast into Mali where it forms a broad inland delta. Beyond the inland delta, the river turns 90 degrees and flows southeast into the Gulf of Guinea of the Atlantic

Ocean. Historically the upper or proto Niger flowed northwest into the Gulf of Senegal during the Pliocene and early Pleistocene (Beadle 1981). This could have allowed faunal exchanges between the two rivers (Senegal and Niger). During the interpluvial phase; dunes advanced southward and blocked off the western route of the Niger River, forcing it northeast towards Timbuktu (Beadle 1981). There was a large inland lake near

Timbuktu that was fed by many of the rivers flowing out of the Sahara during the pluvial periods. At some point a stream capture or break out event drained this large lake following along dune lines to the southeast, forming the course of the Niger River as it is observed today (Talbot 1980).

Little is known about history and past connections of many of the smaller coastal streams. The roughly 21 marine transgression and regression events associated with glaciation that occurred over the last 2.3 million years would have affected these small coastal rivers the most. Low sea level during glacial advances may have exposed former connections and allowed faunal exchanges between the lower courses of adjacent basins.

Conversely, marine transgression during pluvial (interglacial) periods would have flooded the lower courses of coastal rivers and seriously reduced available habitat for primary freshwater fishes. Pluvial periods may have also allowed faunal exchanges to occur by flooding the lower reaches of these rivers, as is hypothesized for the Senegal and Gambie rivers (Roberts 1975), or by causing stream captures among adjacent basins.

Study taxa

Amphiliidae is a family of African freshwater catfishes with 12 genera and 66 species. Currently 28 members of the genus Amphilius inhabit fast moving streams throughout tropical Africa. It has recently been proposed that these species should be split into two groups corresponding to high and low Africa (Thomson 2013). The two groups are easily distinguished by the absence of a crenulated epidermal fold at the base of the caudal fin in the high African taxa and a higher number of principal caudal rays in the high African group (8-9 versus (6-7 or 7-8). All Amphilius spp. from the study area have the crenulated epidermal fold and 6-7 principal caudal rays. If this recommendation were followed the Guinean taxa would retain the genus name, Amphilius, whereas

Anoplopterus Pfeffer, 1889 would be resurrected for the high African taxa (Thomson

2013).

Amphilius species are well-adapted to high gradient Fouta Djallon streams. They have dorsoventrally flattened bodies with reduced eyes, and broad pectoral fins with a stiffened first ray (Figure 1.4). The broad pectoral fins have adhesive microstructures present on the anterior rays (Bell-Cross and Jubb 1973) which allow these fishes to cling to rocky substrate.

Amphilius platychir Gunther, Amphilius atesuensis Boulenger, Amphilius grammatophorus Pellegrin, Amphilius rheophilus Daget, and Amphilius kakrimensis

Teugels et al., are the five species reported to occur within Guinea (Paugy et al. 2003b;

Schmidt & Pezold 2011). Three of these species (Amphilius platychir, A. rheophilus, and

A. atesuensis) are reported as widespread across West Africa, whereas A. kakrimensis is restricted to the Kakrima River (a tributary of the Konkouré River). Recent work has placed the A. platychir populations from Fouta Djallon streams into the resurrected A. grammatophorus Pellegrin, and provided evidence for additional cryptic species within the area (Schmidt and Pezold 2011).

Amphilius platychir is likely restricted to the Little Scarcies, Kolenté, and interior rivers of Sierra Leone. Amphilius grammatophorus is found in the Upper Konkouré,

Gambie, Senegal, and Niger Rivers. Genetically and morphologically distinctive populations in coastal rivers (Fatala and Tinguilinta) of Guinea and Sierra Leone, initially identified as A. platychir, likely represent a new species (Schmidt & Pezold 2011).

Amphilius rheophilus, initially thought to be one widespread species, has been determined to be a complex of species. Amphilius rheophilus is restricted to the Gambie and Rio Corubal rivers; populations from the Senegal and Konkouré, those from the Kaba

River, and those from the Loffa and Loh rivers of southeastern Guinea all represent new

7 species (Schmidt and Pezold 2011). Species of the Amphilius rheophilus complex do not occur in the small coastal rivers of Guinea or Sierra Leone.

The suckermouth catfishes of the genus Chiloglanis are the second most species rich genus within family Mochokidae. There are 49 described species of Chiloglanis found throughout sub-Saharan Africa; with more species awaiting description (Ferraris,

2007). These diminutive (<10 cm) fishes inhabit rivers of all sizes and are typically associated with flowing water (Figure 1.5). Habitats utilized by these fishes vary by species, with many associated with boulders and smaller rock in riffles, while others are found near aquatic vegetation and woody debris.

Members of the genus are distinct from other mochokid genera in possessing an oral disc formed by modified maxillary and mandibular barbels, and in the position and number of mandibular teeth. The disc helps them to maintain position in turbulent flow and aids in feeding. Mandibular teeth in Chiloglanis are usually crowded near the midline of the mouth or spread across the mouth opening in one or two rows (Vigliotta, 2008).

Two oval premaxillary tooth patches, along with buccal suction provided by the disc, hold the fish steady while a row of curved mandibular teeth scrape aufwuchs and other organisms off of rocks, plants, and woody debris (Friel and Vigliotta, 2011). These fish are relatively poor swimmers; they are said to resemble tadpoles once they enter the water column (Daget and Durand, 1981).

Chiloglanis species are found throughout most of the Fouta Djallon highlands and surrounding areas. There are no records of Chiloglanis spp. in the Rio Corubal or Gambie rivers. They also seem to be absent from the lower reaches of the Konkouré River and small coastal river basins. However, there are records of Chiloglanis in the Bofon River

(Figure 1.3) so they may exist in other coastal streams that haven’t yet been thoroughly sampled. There are two species recorded from the Fouta Djallon region and another found in the extreme southeastern part of Guinea. Chiloglanis occidentalis Pellegrin, reported to be widespread throughout West Africa, is found throughout Guinea (Paugy et al. 2003b). A second species of Chiloglanis is recorded from the Fouta Djallon and was found in the upper reaches of the Tinkisso and Niandan rivers(Niger River basin, Daget

1954; Hugueny 1990b). The species was initially identified as Chiloglanis micropogon

Poll, (Daget 1954), but was later synonymized with C. batesii (Roberts 1989). Both of these species were described from Cameroon and display high degrees of sexual dimorphism. Males have an elongated anal fin and a filamentous upper caudal fin lobe.

Recently, C. micropogon was removed from synonymy with C. batesii and recognized as valid (Friel & Vigliotta, 2011). None of the Chiloglanis cf. micropogon specimens collected from the upper Tinkisso River in Guinea display sexual dimorphism and likely represent a new species.

Cyprinidae is a diverse family of freshwater fishes that occurs throughout Africa,

North America, Europe, and Asia (Nelson 2006). More than 800 nominal species of genus Barbus are reported throughout Africa, Asia, and Europe (Berrebi et al. 1996).

Berrebi et al. (1996) recognized 50 nominal species from Europe and northwestern Africa as Barbus s. str. (sensu stricto). It was suggested that all species that could not be placed in Barbus s. str. should be referred to as ‘Barbus’ until they can be placed into appropriate groups (Berrebi & Tsigenopoulos 2003). Current work with ‘Barbus’ in

Africa is aimed at identifying monophyletic groups of species that can be diagnosed as different genera. One example of this is the resurrection of Pseudobarbus Smith, found in

Southern Africa, by Skelton (1988). Another recent change was removing the large hexaploid barbs from ‘Barbus’ and placing them in the resurrected Labeobarbus Ruppëll,

(Tsigenopoulos et al. 2010).

Twenty-eight species of the small diploid ‘Barbus’ and four species of the large

Labeobarbus occur in waters of Guinea and neighboring countries. Some of these species

(e.g.,‘B.’ macrops and ‘B.’ sublineatus) are reported to occur throughout western Africa, while 11 species are endemic to single river basins within the upper Guinea ichthyo- province (Bamba et al. 2011). These species all vary in a combination of the following characters: barbel length and number, meristic counts, and pigmentation patterns

(longitudinal lines, spots, or de-pigmented), and presence or absence of serrations on third dorsal fin ray (Figure 1.6). The taxonomy and distribution of many African

‘Barbus’ is suspect and a full revision is needed (Berrebi & Tsigenopoulos 2003). The small diploid barbs are reported to have invaded Africa from Asia in the early Miocene

(~20 MYa), whereas the large hexaploid barbs are believed to be more recent invaders

(~8MYa) (Tsigenopoulos et al. 2010). Confirming the timing of this invasion will aid in the discovery of recent (Miocene to Pleistocene) dispersal/vicariance events in other groups.

Significance of Proposed Research

The upper Guinean ichthyo-province (Hugueny & Lévêque 1994), which includes coastal rivers from Guinea to Liberia, is one of the least understood faunal provinces in

West Africa (Teugels et al. 1987). One reason for the lack of knowledge of the fish fauna has been the relative paucity of specimens available for study from the area. Recent

10 expeditions (2002-2003, 2013) greatly increased the amount of study material from rivers in the region and also provided tissues samples for genetic research.

The ichthyo-provinces proposed for the area by Hugueny and Lévêque (1994) differ little from those proposed by Roberts (1975). The Gambie River was moved from the Upper Guinean province to the Nilo-Sudan province with the Senegal and Niger

Rivers (Hugueny & Lévêque 1994). The Rio Corubal, Konkouré, Kolenté, Kaba, and rivers within Sierra Leone and Liberia are placed in the Upper Guinean province.

Comparing the above proposed ichthyo-provinces to divergences observed within

Amphilius and the recent discoveries of endemic taxa, it is clear that additional areas of endemism exist within the Fouta Djallon highlands. Morphological evidence coupled with cytochrome b data available for some of the populations suggest the existence of five distinct A. rheophilus lineages (Schmidt and Pezold 2011). Within the Nilo-Sudan province, divergences were observed between the Senegal and Niger River populations, and populations from the Gambie River. Divergences were also observed among populations within the previously defined Upper Guinean province.

A thorough understanding of the diversity and distribution of an area’s fauna is fundamental to the conservation and stewardship of the area. This is especially true for the Fouta Djallon highlands and the forest remnants of southeastern Guinea. This area spans four ecoregions: Fouta Djallon ecoregion, Senegal-Gambia catchments ecoregion,

Southern Upper Guinea ecoregion, and Upper Niger ecoregion (Thieme et al. 2005).

Three of these ecoregions are listed as vulnerable with the Southern Upper Guinea ecoregion listed as endangered. The vulnerable ecoregions have a conservation status of

V while the Southern Upper Region is ranked as IV (Thieme et al. 2005). The rankings

(I-V) determine the conservation priority of an area and are based on biological distinctiveness and conservation status. Class I ecoregions are globally outstanding areas that are highly threatened; class II are continently important and highly threatened; class

III are global or continentally important ecoregions that are not currently threatened; class

IV are low priority bioregionally and nationally important ecoregions that are highly threatened, and class V are bioregionally and nationally important ecoregions that are not presently threatened (Thieme et al. 2005). The ecoregions are based on Roberts’ (1975) ichthyo-provinces with some modifications. As was observed for Amphilius (Schmidt and

Pezold 2011) the biodiversity of other aquatic biota from the region may be underestimated, which would require a reassessment of these rankings.

Guinea has one of the largest reserves of bauxite in the world (Bermúdez-Lugo

2004). In 2004 mineral exports accounted for 80% of the country’s GDP with bauxite being the main export. An estimated 7.5 billion metric tons of bauxite reserves have been identified in Guinea, and are currently producing 17 million metric tons annually

(Bermúdez-Lugo 2004). This mineral, along with iron ore, is extracted using open pit mines which have negative effects on aquatic systems. Artisanal and large scale gold mining operations also pose a significant risk to aquatic communities in Guinea, chiefly from the heavy metal pollution by-products of the mining activities (Donkor et al. 2009;

Nartey et al. 2011).

Another potential threat to the area’s aquatic biodiversity is construction of hydroelectric dams. Guinea has very high (19,400 GWh/year) potential for producing hydroelectric power, and is currently only utilizing 1% of this potential (EIA 2006). This situation will surely change in the future as the country continues to stabilize and foreign

12 investment increases. The Semafo Corporation recently announced plans to construct a

130 MW dam on the Kogon River with construction scheduled to have begun in 2012

(Camara 2011). The Souapiti/Kaleta hydroelectric project is in the early phases of construction. Located on the middle Konkouré River this project will result in the inundation of 780 km2 above the impoundment (Diabete et al. 2008). The dam would limit upstream movement of fish species and reduce the natural flood pulse of the river.

The effects of the Garafiri dam (upper Konkouré River) on fish populations were not clear in the three years after its completion (IRD-BCEOM-BRLi 2003). The Guinea state-owned utility, Electricite de Guinee, announced that it was seeking bids to complete an environmental impact assessment for the dam and its associated fossil-fuel run power plant (Hydroworld [internet]). The reports of this assessment have not yet been made public.

Investigating the biogeography of the Fouta Djallon and surrounding regions may reveal a different history of diversification and additional areas of endemism than previously hypothesized, as was suggested by a recent study involving two Amphilius species complex (Schmidt and Pezold 2011). A comparative phylogeography study involving multiple co-distributed species complexes with multiple genetic loci may establish a general pattern of vicariance for the region. The biogeographic units proposed by this study could then be used to aid biodiversity discovery and conservation of other elements of the aquatic biota.

Hypotheses and Tests

The currently accepted view of diversification within the Fouta Djallon region is primarily based on faunal inventories (Hugueny and Lévêque 1994). These faunal

13 inventories were derived from checklists of individual drainages in the area (Lévêque et al. 1989; 1991; Paugy & Bench 1989; Paugy et al. 1990; Teugels et al. 1988) and the

Check-list Of the Freshwater Fishes of Africa (Daget et al. 1984; 1986a; 1986b; 1990).

Although these checklists represent a large advancement in the understanding of African freshwater fish taxonomy, they no longer adequately describe the diversity and distribution of species within Africa. Since 1990, around 450 new species of fish have been described across Africa (Skelton and Swartz 2011). The first step in elucidating the historical biogeography of a region is to understand the diversity and distribution of the species present.

A study of the Amphilius of the Fouta Djallon region and surrounding areas discovered more diversity than was previously reported (Schmidt and Pezold 2011).

Amphilius platychir was reported to be widespread throughout West Africa (Daget et al.

1986a); however, within the study area alone, A. platychir was found to comprise three distinct species (Schmidt and Pezold 2011). Amphilius rheophilus was also reported to be a single widespread species within the area, but Schmidt and Pezold (2011) discovered five distinct populations that likely represent distinct species. No studies of genetic or morphological variation have been conducted on Chiloglanis populations from the area.

Since A. rheophilus and Chiloglanis species occur sympatrically over much of the Fouta

Djallon and occupy similar ecological niches; I hypothesize that a molecular and morphological analysis of the group will result in the discovery of additional Chiloglanis taxa, likely exhibing a similar pattern of diversification over a similar time frame.

Consistent with this hypothesis, recent studies of ‘Barbus’ species from the area

(Lévêque et al. 1988; Bigorné and Lévêque 1993; Bamba et al. 2011) have resulted in the

14 discovery of new taxa and have refined understanding of the distribution of ‘Barbus’ spp. from the area. I hypothesize that a molecular and morphological analysis of select‘Barbus’ lineages from the area will result in the discovery of additional new taxa and will further change understanding of distributions of ‘Barbus’ species from these lineages.

Previous biogeographic hypotheses for the Fouta Djallon region have been based upon faunal inventories because the paleohydrology for much of the area is not well known. The history of African river systems since the Cretaceous has been examined only in broad geographic terms and provides little information of relevance to the present study (Beadle 1981; Lévêque 1997; Goudie 2005). As detailed earlier, hydrological research has been conducted on the Senegal and Gambie rivers (Michel 1973). Other research has examined the climatic variability of sub-Saharan West Africa during the

Holocene and late Pleistocene utilizing palynological and sedimentary evidence

(Livingstone 1975; Frédoux 1994; Coe 1997; Gasse 2000; Verschuren 2003; Drézen et al. 2010). This research details the successive periods of moisture and drought across

West Africa, but does not address the possible paleodrainage connections among many rivers in the area. It also does not detail or hypothesize hydrologic events old enough to account for the divergence of populations into new species.

Currently only two hypotheses of paleohydrological conditions of the Fouta

Djalon region can be constructed from existing evidence. The first is that the upper Niger

River flowed into the Senegal River until the Pleistocene when dry conditions caused the

Niger to flow northeast (Beadle 1981). Considering this, I hypothesize that populations of

Amphilius, Chiloglanis, and ‘Barbus’ species from the Senegal and upper Niger rivers

15 should exhibit little divergence and that any divergence observed between populations from the two rivers would be a result of isolation during the late Pleistocene (~40,000

BP). Second, the Senegal and Gambie rivers remained isolated from each other until the

Pleistocene when dry conditions caused the Senegal River to flow into the Gambie River

(Michel 1973). Also, during pluvial phases of the Pleistocene, faunal exchanges could have occurred between the lower reaches of these rivers (Roberts 1975). From this, I would expect to observe genetic divergence among populations of Amphilius,

Chiloglanis, and ‘Barbus’ species from the Senegal and Gambie Rivers owing to pre-

Pleistocene isolation. However, more recent (Pleistocene) dispersal events may have reduced some of this divergence.

The paleohydrology of the westward flowing rivers within the Fouta Djallon region is unknown. Studies involving other fish taxa have used historical connections among populations to aid in the discovery of paleohydrologic events (Smith and Dowling

2008; Schönhuth et al. 2011). Divergences observed among populations can be used to estimate the timing of divergence events using molecular clock techniques (Betancur-R and Armbruster 2009). These estimates can then form the basis of a general hypothesis of paleohydrologic evolution in the area of interest. The fluctuations in sea level that occurred during the Pleistocene would have restricted available habitat and limited dispersal routes for primary freshwater fishes within the lower reaches of coastal rivers, but the extent to which this occurred is unknown. Based on available zoogeographic evidence, which is dependent on admittedly outdated taxonomy, the most logical null expectation would be that populations from coastal plain reaches of these westward flowing (coastal) rivers will show little genetic structure among river basins. An

16 alternative hypothesis would be that populations within the coastal rivers will show genetic differences as a result of physical isolation among the basins.

The headwater reaches of the rivers in West Africa have been relatively stable since the middle Miocene (Lévêque 1997). If the headwaters of westward flowing rivers conform to this pattern of relative isolation, one would expect to observe genetic divergence of populations from different rivers basins. Alternatively, past headwater capture events (biotic dispersal), or dispersal among the lowland reaches of the rivers and subsequent dispersal upstream would tend to make headwater populations genetically uniform. Phylogenetic analysis coupled with molecular clock techniques may allow estimation of the timing of these events and the extent to which headwater capture or lowland dispersal played a role in the observed diversity (Burridge et al. 2006).

Including species complexes from two different orders of fishes (Cypriniformes and Siluriformes) in this analysis increases the probability that generalized biogeographical patterns for the Fouta Djallon region will be discovered. The two

Siluriformes families included are endemic to Africa and are believed to have originated in Central Africa. A hypothesis of pan-African biogeography can be advanced by including specimens representing species from the two catfish families collected in

Central and East Africa in the analysis. The barbs are European in origin and are believed to have invaded northeast Africa 20 MYA (Tsigenopoulos et al. 2010). The pathway of cyprinid dispersal across tropical Africa is unknown. Including cyprinids from other parts of Africa will allow me to develop hypotheses of pan-African cyprinid biogeography.

This study aims to construct a general hypothesis of historical biogeography for the Fouta Djallon and surrounding areas by completing phylogeographic analyses of

17 species complexes from three co-distributed groups of fishes (Amphilius, Chiloglanis, and Barbus) from the region. The resulting general biogeographic hypothesis can then be utilized by researchers examining the biodiversity of other taxa in the area. The new fish taxa discovered through this work can be added to existing faunal inventories allowing the reassessment of present conservation priorities. The outcomes of this research, added to the patchwork of research occurring throughout Africa, should aid in developing some pan-African biogeographical hypotheses.

Figure 1.1. The rivers of Africa with demarcation between High and Low Africa. The study area, Guinea, outlined in black.

Figure 1.3. Rivers of the Fouta Djallon highlands and surrounding areas. Generalized extent of Fouta Djallon highlands denoted by dotted-line. Extent of the three proposed ichthyological provinces (Upper Guinean, Nilo-Sudan, and Eburneo-Ghanean) within dashed line marking the boundaries.

Figure 1.4. Amphilius cf. platychir from Fouta Djallon (Badi River, bar = 1 cm). Note the dorsal-ventrally compressed profile and broad stiffened pectoral fins.

Figure 1.5. Chiloglanis cf. occidentalis from the Fouta Djallon (Senegal River basin, bar

= 1 cm). Note the distinctive oral disc, dorsal-ventrally compressed body, and spines in dorsal and pectoral fins.

(note the absence of barbels and the incomplete lateral line, bar = 1cm).

CHAPTER 2

PHYLOGEOGRAPHY OF THE SUCKERMOUTH CATFISHES (MOCHOKIDAE:

CHILOGLANIS) FROM THE FOUTA DJALLON HIGHLANDS AND

SURROUNDING AREAS.

Abstract

Members of the genus Chiloglanis occur throughout tropical Africa. Recent studies of

Chiloglanis revealed previously unrecognized diversity within the group. This study investigates genetic variation in three Chiloglanis species currently recognized from

Guinea, West Africa. The study is based on specimens and tissues samples collected during expeditions in 2003 and 2013. Here I present a phylogeography of Guinean

Chiloglanis populations inferred from mitochondrial (Cyt b) and nuclear (Growth

Hormone intron) markers. The study revealed numerous new species that require formal description. The study also confirmed the presence of Chiloglanis cf. micropogon within the upper Niger River basin, which resolves as sister to C. micropogon specimens from the Congo. The phylogeography of Chiloglanis from Guinea is discussed and preliminary hypotheses of the historical biogeography of the area are presented.

Introduction

The Fouta Djallon highlands of Guinea are believed to serve as an important biogeographic barrier in West Africa (Lévêque 1997). These highlands, rising over 1500 meters, separate two recognized ichthyo-provinces. Rivers draining the western slopes of the Fouta Djallon are within the Upper Guinean province, and rivers on the northern and

25 eastern slopes are within the Nilo-Sudan province (Hugueny & Lévêque, 1994, Figure

2.1). While the highlands appear to serve as an effective dispersal barrier for most taxa, certain taxa are reported to occur on both sides of the Guinean range (Daget 1962). These so-called “amphi-Guinean” taxa include species of Amphilius, Kribia, Chiloglanis, and rheophilic barbs (‘Barbus’). A recent study investigating Amphilius found additional diversity within the Guinean highlands and called into question the presence of these amphi-Guinean taxa (Schmidt & Pezold 2011). Here I investigate the phylogeography of another proposed amphi-Guinean group, the suckermouth catfishes of the genus

Chiloglanis.

Suckermouth catfishes are the second most species rich genus within the family

Mochokidae. Forty-nine described species of Chiloglanis occur throughout sub-Saharan

Africa (Ferraris, 2007). These diminutive (<10 cm) fishes inhabit rivers of all sizes and are typically associated with flowing water. Members of the genus differ from other mochokid genera in the position and number of mandibular teeth, and in possessing an oral disc formed by modified maxillary and mandibular barbels. The disc helps them to maintain position in turbulent flow and aids in feeding. Mandibular teeth in Chiloglanis are usually crowded near the midline of the mouth or spread across the mouth opening in one or two rows (Vigliotta, 2008). Although poor swimmers when in the water column

(Daget & Durand 1981), the presence of the oral disc may allow Chiloglanis to migrate upstream or downstream by crawling over hard substrates.

Three species of Chiloglanis are reported to occur within Guinea (Daget et al.

1986). Chiloglanis occidentalis Pellegrin 1933 is a widespread species described from the

Sassandra River in Côte d’Ivoire. Chiloglanis cf. micropogon is found in the upper

26 reaches of the Tinkisso and Milo rivers (Niger R. basin). These fishes are thought to be related to C. micropogon Poll 1952 from the Congo River basin. Lastly, C. lamottei

Daget 1948 was described from a single specimen collected near Mt. Nimba in streams draining into the Cavally River. Additional specimens of this species have only recently been collected near Mt. Nimba during the 2013 expedition. Expeditions in the region during 2003 sampled specimens from populations of C. occidentalis throughout Guinea, including both sides of the Fouta Djallon. A preliminary assessment of these specimens revealed significant morphological variation among populations. The expedition in 2013 added to these collections and provided much needed genetic material for C. occidentalis,

C. cf. micropogon, and C. lamottei populations. Mitochondrial and nuclear markers were sequenced for all sampled populations of all three species to investigate the phylogeography of Guinean Chiloglanis. By comparing the results of this study to results for other co-distributed taxa from the area, a general historical biogeography can be advanced for the Fouta Djallon highlands and surrounding areas.

Methods and methods

Taxon sampling and protocols

Chiloglanis specimens were collected at 61 localities within Guinea during four expeditions (Figure 2.2). Specimens were collected by seine, dip net, and electrofisher.

Chiloglanis tissue samples were collected at 27 of these localities (Figure 2.2). Tissue samples were stored in 95% ethanol and voucher specimens were photographed if possible. Vouchers and other collected specimens were then fixed in 10% formalin and later transferred to ethanol or isopropanol. After identification, specimens will eventually be deposited in AMNH, AU, CUMV, and TU. Tissue samples and other genetic material

27 were deposited at genetic resources collections for long term storage. In addition to material collected from Guinea, material from East and Central Africa were included in the analysis. All specimens included in the molecular analysis are listed in Table 2.1.

Total DNA was extracted from preserved muscle and/or fin tissue using an

Invitrogen PureLink Genomic DNA mini prep kit. The mitochondrial cytochrome b gene and intron 4 of the nuclear Growth Hormone (GH) were used to infer the phylogenetic relationships of Guinean Chiloglanis. Cytochrome b is widely used to investigate species level phylogenies. The cytochrome b region was amplified using the primers Glu-2 and

Pro-R1 (Hardman and Page 2003) with a touchdown protocol described in Schmidt et al.

(in review). GH introns have been used to infer the phylogeny of North American minnows (Cyprinidae: Hybognathus, Moyer et al. 2009), and small diploid barbs from

Kenya (Cyprinidae: ‘Barbus’, unpublished data). A recent study demonstrated the utility of intron markers for resolving relationships of Kenyan species of Chiloglanis (Schmidt et al. in review). The GH intron 4 was amplified for representative individuals from each population of Chiloglanis using previously established primers and protocols (Schmidt et al. in review).

Amplified products were confirmed by visualization on an Ethidium Bromide stained gel. Fragments were purified with ExoSAP-IT (Amersham Biosciences) and sequenced with the amplification primers using the BigDye Terminator Reactioin Mix from Applied Biosystems. Sequencing reactions were visualized on an ABI 3730XL

DNA analyzer and contigs were aligned and visually edited in Seqeuncher 4.6 (Gene

Codes Corporation). All products were submitted to GenBank. Accession numbers from each product are listed in Table 2.1.

Sequence alignment and phylogenetic analyses

The partial cytochrome b sequences were aligned with ClustalW v 1.4 (Thompson et al.

1994) and edited in BioEdit v. 7.0.9 (Hall, 1997). Intron 4 sequences were aligned with

Prankster (Löytynoja and Goldman, 2008). The indels (ambiguous areas within alignment) were coded in FastGap version 1.2 (Borchsenius, 2009) using the simple coding method (Simmons and Ochoterena, 2000). The characters derived from coding the indels were then concatenated to the end of the intron 4 alignment. From these alignments two analyses were completed. The first alignment was just the cytochrome b data; the second, a partitioned analysis, consisting of cyt b, GH intron 4, and the gap characters derived from gap coding.

MrModeltest 2.0 was used to determine the optimal model of evolution for the cytochrome b and GH intron 4 alignments. Maximum likelihood (ML) and Bayesian inferences analyses were performed to infer the phylogeny. GARLI 2.0 (Zwickl, 2006) via the CIPRES Science Gateway (Miller et al. 2010) was used to perform the ML analyses. Nodal support was inferred by bootstrap proportions after 1000 bootstrap replicates with 3 search reps each. Bootstrap values were obtained by producing majority rule consensus trees in Mesquite (Maddison and Maddison, 2011). Bayesian analysis was performed on the two datasets in MrBayes version 3.2 (Rohnquist et al. 2011). Posterior probabilities were assessed with 5 million generations, sampling trees every 100 generations. The first 25% of trees were discarded as burn-in.

Divergence estimation

A concatenated dataset of cytochrome b and GH intron 4 was used to estimate the divergence times of Guinean Chiloglanis populations, as implemented in BEAST version

1.4.2 (Drummond et al. 2006). A lognormal relaxed clock rate variation model and birth- death speciation process was used as a tree prior. The most recent common ancestor

(MRCA) for the study group was set at 24-6 Ma. The calibration point was derived from recently published studies on African mochokids (Day et al. 2013). In addition to this imprecise calibration point, divergence estimates (ucld.mean) for each marker were set as uniform priors (Cyt b .76-2.0, GH intron 4 0.001-.33). Substitution rates of Cyt b are well known for teleost fishes (Chakona et al. 2013). Rates of GH introns are estimated from rates observed in East African Chiloglanis (Schmidt et al. in review).

In Bayesian analyses, Markov chains were run for 80 million generations and sampled every 1000. Resulting log files were analyzed with Tracer 1.4 (Rambaut and

Drummond, 2007) to ensure that all effective sample sizes were above 100. Resulting trees were resampled to reduce the number of trees to 40,000, after which 10,000 were discarded as burn-in. Final tree and 95% highest posterior distributions were visualized in

FigTree v 1.4 (Rambaut 2007).

Results

Cytochrome b and GH intron 4

An 1108 base pair (bp) alignment of partial cyt b was obtained for 83 Chiloglanis specimens from across Guinea, the DRC, and Kenya. Specimens from Kenya were designated as outgroups. Parsimony informative (PI) characters in the alignment numbered 322. Uncorrected pairwise distances ranged from 14% - 18% among ingroup and outgroup taxa. Pairwise distances among ingroup taxa varied from 2% - 13%. The model of evolution selected by the AIC and implemented in the ML and Bayesian analysis was TVM + I + G (0 1 2 3 1 4).

A majority-rule consensus tree obtained from Bayesian inference and ML analyses revealed the presence of numerous monophyletic groups of Chiloglanis spp. within Guinea (Figure 2.4). All branches were well-supported with high posterior probabilities (pp) in the Bayesian tree; whereas, some branches (shown in gray) were poorly supported and would be collapsed in the ML tree. Branch support values and uncorrected average distance between clades are shown in Figure 2.4. The Chiloglanis micropogon group is basal and sister to all other Guinean taxa. Chiloglanis cf. micropogon specimens collected in the upper Niger River drainage are sister to specimens from the Democratic Republic of the Congo (DRC) with an uncorrected p- distance of 3.7% between them.

Chiloglanis lamottei is sister to remaining Guinean taxa with moderate bootstrap

(71) and pp (.82) support. Chiloglanis sp. “Loffa sp. 2” is sister to other Chiloglanis taxa and is well-supported in the Bayesian analysis. However, this branch is poorly supported and collapses in the ML analysis, resulting in an unresolved polytomy involving Loffa sp.

2 and two other groups of taxa designated as “short spine” and “long spine” groups.

Specimens in the “short spine” group have shorter dorsal and pectoral spines than those in the “long spine” group. The “short spine” group comprises specimens from the

Konkouré/Badi, Little Scarcies/Kolenté, and Moa Rivers; whereas the “long spine” group comprises specimens from the Senegal/Niger, Loffa (sp. 1), St. John, and St. Paul River populations (Figure 2.4).

Within the “short spine” group, specimens from the Moa (Chiloglanis sp. “Moa

R.”) are the basal sister group to the rest of the group. Specimens from the Little Scarcies

(Chiloganis sp. “Little Scarcies”) are sister to specimens from the Kolenté River, and this

31 group is sister to specimens from the Konkouré and Badi Rivers (Figure 2.4). “Long spine” group specimens from the Niger and Senegal rivers are sister to a group of taxa from the Loffa, St. Paul, and St. John rivers. This well-supported branch collapses in the

ML analyses. Species from the St. John River (Chiloglanis sp. “St. John”) are sister to

Chiloglanis sp. “Loffa” and Chiloglanis sp. “St. Paul”.

A concatenated dataset of 1,680 characters was obtained for 40 representative individuals from all groups discovered in the Cyt b analysis. The dataset included the Cyt b alignment (1108 bp), Prankster alignment of GH intron 4 (549 bp), and the characters derived from gap coding GH intron 4 (23 characters). There were 35 PI characters in the

GH intron 4 alignment and 17 PI characters in the gap coded characters. The model of evolution selected by the AIC for the GH intron was TPM3 uf + G (0 1 2 0 1 2). The gap characters were treated as standard binary characters. The phylogeny inferred from this concatenated dataset (Figure 2.4) is largely congruent with that resulting from the Cyt b analysis. The topology is identical; however, additional branches lack bootstrap support in the ML analysis.

Divergence estimation

The inferred age of the most common recent ancestor between the C. micropogon group and the remaining ingroup taxa was 13.5 Ma (24 – 6 Ma set as prior, Figure 2.5). The mean age for the split between C. lamottei and the remaining Guinean taxa was 10.3 Ma

(95% Highest Posterior Distribution (HPD): 18.2 – 5.7). Chiloglanis sp. “Loffa sp. 2” diversified from the “short spine” and “long spine” groups around 7.6 Ma (95% HPD:

13.2 – 3.9) and the latter two groups diverged around 6.7 Ma (95% HPD: 11.8 – 3.5).

Within the “short spine” group, Moa River populations diverged from the remaining taxa

4.1 Ma (95% HPD: 7.7 – 1.7). Divergence of the Konkouré/Badi river and Little

Scarcies/Kolenté river populations occurred around 2.9 Ma (95% HPD: 5.6-1.1).

Divergences within each of these groups is recent (1.3 Ma, 95% HPD: 2.8 – 0.4 between the Badi River and Konkouré River and 1.1 Ma, 95% HPD: 2.7 – 0.4 between the

Kolenté River and the Little Scarcies). Chiloglanis sp. “Senegal/Niger” diverged from other “long spine” taxa 5.6 Ma (95% HPD: 9.8 – 2.9). Chiloglanis sp. “St. John” diverged from the Chiloglanis sp. “St. Paul” and Chiloglanis sp. “Loffa” around 4.1 Ma

(95% HPD: 7.4 – 1.9). The age of the split between C. sp. “St. Paul” and C. sp. “Loffa” is approximately 2.0 Ma (95% HPD: 4.1 – 0.7).

Discussion

Undescribed Chiloglanis taxa within Guinea

This study confirmed the presence of multiple additional species within the taxon currently recognized as Chiloglanis occidentalis. The genetic divergences within Cyt b and GH intron 4 correspond to observed morphological variation (Figure 2.6). These new species are largely allopatric in their distributions across Guinea (Figure 2.7). Chiloglanis sp. “Loffa sp. 2” is the basal sister group and represents a new species. Within the “short spine” group are newly discovered species in the Moa River, Konkouré/Badi rivers, and

Little Scarcies/Kolenté rivers. A morphological study of these specimens is ongoing and will determine if the recently diverged populations in the Konkouré and Badi rivers and those in the Little Scarcies and Kolenté River are distinct taxa requiring formal description. Four new species are evident within the “long spine” group. These are

Chiloglanis sp. “Senegal/Niger”, Chiloglanis sp. “Loffa”, Chiloglanis sp. “St. Paul”, and

Chiloglanis sp. “St. John”.

Recognizing all of these groups as new taxa requires renewed circumscription of

Chiloglanis occidentalis. Described from the Sassandra River in Cote d’Ivoire, this species range, within Guinea, is likely confined to the Bafing R. (Sassandra R. drainage) in southeastern Guinea. Chiloglanis specimens were not collected in the two Bafing R. localities sampled during the 2013 expedition. A complete morphological study of all

Chiloglanis taxa from Guinea and topotypic C. occidentalis material is ongoing and will result in a systematic revision along with formal descriptions of all new taxa discovered herein.

The sister relationship between C. cf. micropogon specimens from the upper

Niger River drainage and C. micropogon specimens from the DRC confirms Daget’s

(1954) determination that a C. micropogon-like species is in the upper Niger and raises some interesting biogeographical questions. The genetic divergence and morphological variation observed between these groups likely warrants specific recognition for the upper Niger R. populations. In additional to confirming the presence of C. cf. micropogon, this study also substantiates the presence of C. lamottei within the Cavally

River in southeastern Guinea. This species was only known from the holotype and was based on a rather vague description (Daget 1948). The genetic material and additional specimens collected during the 2013 expedition will allow redescription of C. lamottei and formal description of C. cf. micropogon.

Historical biogeography of Guinean Chiloglanis spp.

The phylogeny presented herein is the first of its kind produced from the area. As

Chiloglanis spp. are distributed throughout much of the study area; the inferred relationships provide novel insights into the history of Chiloglanis diversification in the

34 region, and suggests past hydrological connections across the Fouta Djallon and surrounding areas. The paleohydrology of rivers on the western slope of the Guinean range is poorly known; however, some reconstructions of the Niger, Senegal, and

Gambie rivers have been completed. The age of the area is fairly well documented

(Michel 1973; Lévêque 1997). The Guinean Range first arose in the Late Jurassic (~106

– 104 Ma) and again in the upper Eocene (~38 – 34 Ma). Since this time the region has been relatively stable with ongoing erosion. More recently (40000 BP), rivers became further incised with increased rainfall (Michel 1973).

The most basal Chiloglanis of the study area is C. cf. micropogon, which diverged from C. micropogon from the eastern Congo around 1.4 Ma (3.5 – 0.2). This taxon is likely a remnant of a more widespread ancestor that colonized the upper reaches of the

Niger River system and subsequently diverged. The Benue River (lower Niger River) lies in close proximity to headwater streams of the eastern Congo. This proximity could have allowed faunal exchange between the two systems.

Results of the BEAST analysis suggests that Chiloglanis lamottei diverged 10.3

Ma from the MRCA of the clade represented by Chiloglanis sp. “Loffa sp. 2” plus the long and short spined Chiloglanis. Another vicariant event occurred around 7.6 Ma that gave rise to Chiloglanis sp. “Loffa sp. 2” and the MRCA for all the remaining

Chiloglanis taxa from the area. This ancestor diverged into clades representing the “short spine” and “long spine” species groups around 6.7 Ma (Figure 2.5).

The basal most taxon in the “short spine” group is represented by the species from the Moa River, which diverged from the MRCA of other “short spine” taxa, descendants of which are presently found in the Konkouré and Badi river systems and the Kolenté and

Little Scarcies river system, around 4.1 Ma. Unfortunately, it was not possible to collect specimens from the rivers of Sierra Leone that lie between the Moa R. and Little Scarcies basin in Guinea. One could hypothesize that populations occurring in westward flowing rivers between the Moa R. and Little Scarcies would show affinities with the “short spine” group. The upper reaches of the Bagbe River (Sewa R. Basin, Figure 2.1, 2.2) contains C. polyodon Norman, 1932, a species only known from the type specimen. The type specimen appears to have shorter spines and mental barbels, which would suggest an affinity with the “short spine” group. Specimens from this river along with the upper reaches of the Jong and Rokel river basins would provide additional information on the biogeography of the “short spine” group.

The “short spine” Chiloglanis taxa presently occupying the Little Scarcies and

Kolenté river basins diverged from taxa in the Badi River and the Konkouré River proper around 2.9 Ma (Figure 2.5, 2.7). The short-spine form in the Little Scarcies basin diverged from the form in the Kolenté 1.2 MYA. The “short spine” form in the Badi

River, a lower tributary to the Konkouré River, diverged from the common ancestor of other upper Konkouré River populations around 1.3 Ma (Figure 2.5). A few processes could explain the relatively shallow divergence between the Konkouré and Badi rivers. A wide spread species distributed throughout the Konkouré and Badi rivers could have become isolated in the upper reaches of the two rivers due to a marine transgression event during the Pleistocene. Also headwater capture (biotic dispersal) between the Badi and upper Konkouré River tributaries (e.g. Mayonkouré R.) could have introduced the common ancestor to the Badi, resulting in the sister species relationship observed in this study.

The Chiloglanis population in the Bofon River provides a means of assessing the likelihood off Chiloglanis spp. dispersing among coastal plain rivers versus via headwater capture. The Bofon River’s headwaters are separated by a short distance from the Badi River and the mouth of the Bofon enters the Atlantic Ocean about 25 km north of the mouth of the Kolenté River. If Chiloglanis populations from the Bofon are more closely related to the Kolenté River than the Badi River, this would support dispersal via coastal inundation. Unfortunately, samples taken from the Bofon in 2013 failed to produce any Chiloglanis specimens.

The “long spine” group diverged from the “short spine” Chiloglanis around 6.7

Ma; the long-spine group subsequently diverged into groups on the eastern slopes of the ridge (Niger River basin) and the western slope (St. Paul, St. John, and Loffa River).

Once in the Niger River, Chiloglanis dispersed quickly throughout the upper Niger and

Senegal River basins. The very low levels of genetic structure observed among populations in the Niger and Senegal Rivers suggests that exchanges between these basins are ongoing or occurred very recently.

Among the “long spine” taxa on the western slope of the Guinean range,

Chiloglanis sp. “St. John” was the first to diverge. The St. Paul River and Loffa River taxa diverged around 2.0 Ma (Figure 2.5). This divergence was caused by the isolation of the two forms once the common ancestor established populations in the two rivers.

Faunal exchange between these two rivers through headwater capture could have caused this observed divergence. The event caused secondary contact between Chiloglanis sp.

“Loffa” and the more basal Chiloglanis sp. “Loffa sp. 2”.

Within the Loffa River basin is just one of a number of cases where Chiloglanis species from different clades occur sympatrically. Within the upper reaches of the Niger

River basin, C. cf. micropogon and Chiloglanis sp. “Niger/Senegal” co-occur. Also within the Moa River a species representing the “long spine” clade was collected alongside a species from the “short spine” clade (Figure 2.6 E and M). The co-occurring species appear to be utilizing different habitats. The more diminutive, “short spine” taxa inhabit riffles with medium to large gravel substrates. The “long spine” taxa tend to occur adjacent to the flowing areas and are usually associated with woody debris.

The hypothesis that Chiloglanis occidentalis is an amphi-Guinean taxon, i.e., occurs on both slopes of the Fouta Djallon (Daget 1962) is not supported by the results of this study. Chiloglanis occidentalis is likely restricted to the Sassandra R. basin; three new taxa occur within the Fouta Djallon highlands. Chiloglanis sp. “Senegal/Niger” occurs on the eastern slope, Chiloglanis sp. “Konkouré” inhabits the western slope, and

Chiloglanis sp. “Little Scarcies” is found on the southern slope (Figure 2.7). Chiloglanis sp. “Senegal/Niger” is distantly related to the western and southern slope taxa, diverging from a common ancestor 6.7 Ma (95% HPD: 11.8 – 3.5). The western and southern slope species are sister taxa and diverged more recently, around 2.9 Ma (5.6-1.1).

Conclusions

This study revealed the existence of several divergent Chiloglanis clades within the previously thought widespread C. occidentalis, and confirmed the presence of C. lamottei and C. cf. micropogon in Guinea. Although a morphological study of these taxa is ongoing, numerous new species require formal description. In addition to the new species discovered; novel insights into the biogeography of the area are presented. First

38 and foremost, it does appear that the Fouta Djallon highlands currently serve as a barrier to range expansion for Chiloglanis taxa on the eastern and western slopes of the Guinean range. These species are not sister taxa, and they have different histories on the two sides of the range. How these taxa came to inhabit the Fouta Djallon highlands also impacted their ability to expand within the region. Chiloglanis likely colonized the Konkouré River from the South, resulting in the current distribution where no Chiloglanis occur upstream of the major waterfalls within this basin (Figure 2.1). Further south, the eroding Guinean range has allowed range expansion (headwater capture) and subsequent diversification of taxa on either side of the ridge. There is a general pattern of Chiloglanis taxa moving south to north as dispersal events allow (Figure 2.7).

The pattern of diversification revealed within Chiloglanis superficially corresponds to the proposed ichthyo-provinces; however, the proposed provinces fail to adequately convey either the level of diversification that has occurred within regions or the relationships among regions. The results of this study show that the history of the area is much more complex and dynamic than previously thought. The examination of other widespread taxa from the area will help to elucidate the area’s complex history.

Table 2.1 cont. Chiloglanis specimens included in the molecular analysis (species name, collection locality, voucher No or field No, GenBank accession No for Cyt b, GenBank accession No for GH intron 4. Specimens will be associated with field numbers when deposited at genetic repositories.

Figure 2.1. Rivers of the Fouta Djallon highlands and surrounding areas. Generalized extent of Fouta Djallon highlands denoted by dotted-line. Extent of the three proposed ichthyological provinces (Upper Guinean, Nilo-Sudan, and Eburneo-Ghanean) within dashed line marking the boundaries.

Figure 2.2. Localities (open circles) where Chiloglanis specimens (filled circle) and tissue samples (star) were collected during the 2003 and 2013 expeditions. Extent of proposed ichthyological provinces denoted by dashed lines and water falls indicated by hatched lines.

Figure 2.4. Phylogeny of Chiloglanis spp. inferred from concatenated (Cyt b, GH intron

4, and character derived from indels) dataset with posterior probabilities above and bootstrap support (bs) below (> 95 and .95 represented by *). Gray branches (bs <50) collapse in maximum likelihood analysis.

Figure 2.5. Chronogram of Chiloglanis spp. inferred from BEAST analysis of concatenated dataset (Cyt b and GH intron 4). Estimated age (millions of years) is given and gray bars represent 95% confidence intervals (HPD: Highest Posterior Distributions).

Figure 2.6. Lateral and ventral images of representative morphotypes from each

Chiloglanis species identified in this study (scale bar = 1 cm). A) Chiloglanis sp.

“Konkouré” and B) Chiloglanis sp. “Badi”.