Insight Into the Diversity of the Genus Coptodon

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Cichlids of the Banc d'Arguin National Park, Mauritania: Insight into the diversity of the genus Coptodon

Article in Journal of Fish Biology · February 2016 DOI: 10.1111/jfb.12899

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Cichlids of the Banc d’Arguin National Park, Mauritania: insight into the diversity of the genus Coptodon

N. G. Kide*, A. Dunz†, J.-F. Agnèse‡, J. Dilyte§, A. Pariselle‡‖, C. Carneiro¶, E. Correia¶, J. C. Brito§, L. O. Yarba¶, Y. Kone** and J.-D. Durand§§ ‡‡

*Université Abdel Maleck Essaâdi - Faculté des Sciences, Département de Biologie, Laboratoire Biologie Appliqué et Pathologie, Tétouan, Morocco, †Bavarian State Collection of Zoology, Department of Ichthyology, Münchhausenstr. 21, 81247 München, Germany, ‡Institut de Recherche pour le Développement (IRD), Université Montpellier, UMR 226, Institut des Sciences de l’Evolution de Montpellier, Place Eugène Bataillon, CC 65, F-34095, Montpellier cedex 5, France, §CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto Instituto de Ciências Agrárias de Vairão, R. Padre Armando Quintas, 4485-661 Vairão, Portugal, ¶Observatoire du Parc National du Banc d’Arguin, Nouakchott, Mauritania, ‖IRD, ISE-M, BP 1857 Yaoundé, Cameroon, **Faculté des Sciences de l’Université de Nouakchott, Nouakchott, Mauritania and §§Institut de recherche pour le développement (IRD), Université Montpellier, UMR MARBEC, Place Eugène Bataillon, CC 93, F-34095 Montpellier cedex 5, France

(Received 28 May 2015, Accepted 17 December 2015)

To determine the species diversity of cichlids in the Banc d’Arguin National Park (PNBA) and their phylogenetic relationships with other species in West Africa, a morphometric and meristic and molecular phylogenetic study was conducted. Both approaches not only confirm the presence of Sarotherodon melanotheron in PNBA but also demonstrate the presence of a second species from the genus Coptodon. While morphometric characteristics match the description of the Guinean tilapia Coptodon guineensis, phylogenetic reconstructions based on three mitochondrial and one nuclear DNA fragment demonstrate that C. guineensis is paraphyletic over its range. Because different lineages of C. guineensis are allopatric, the distribution of C. guineensis should be restricted to Ghana and Côte d’Ivoire. The many other lineages of this species should be considered as C. sp. aff. guineensis. © 2016 The Fisheries Society of the British Isles

Key words: Cichlidae; cryptic species; phylogeny; West Africa.

INTRODUCTION Cichlidae is one of the most speciose families of vertebrates, found in South and Central America, Madagascar, Africa, The Middle East, India and Sri Lanka (Kocher, 2004; Salzburger & Meyer, 2004). As with many other families of fishes, cichlid taxonomy is debated because systematics have been profoundly questioned and secondarily revised

‡‡Author to whom correspondence should be addressed. Tel.: + 84 1 223747334; email: Jean-Dominique. [email protected] 1

© 2016 The Fisheries Society of the British Isles 2 N. G. KIDE ET AL. with information from molecular phylogenetics (Chakrabarty, 2010; Dunz, 2012). Global cichlid diversity consists of major lineages with phylogenetic relationships consistent with Gondwanan break-up (Zardoya et al., 1996; Streelman et al., 1998; Sparks & Smith, 2004; McMahan et al., 2013). All African cichlid diversity belongs to the subfamily Pseudocrenilabrinae Fowler 1934 and includes the haplotilapiine formerly referred to as ‘Tilapia’ (Schwarzer et al., 2009; Dunz & Schliewen, 2013). The most recent comprehensive molecular phylogenetic review demonstrated that the haplotilapiine lineage is monophyletic and consists of 10 clades, with all East African cichlid tribes being recovered in a single clade (Dunz & Schliewen, 2013). Considering the molecular phylogeny and some diagnostic morphometric characteristics, Dunz & Schliewen (2013) proposed a novel generic and supra-generic classification of haplotilapiine. In this new classification, the tribes Oreochromini and Coptodonini harbour most of the genus and species diversity of West African cichlids. The extent of species diversity, however, remains to be evaluated because many new species are still being described (Dunz & Schliewen, 2010a, b; Dunz, 2012). Similar to the East African Great Lakes, Cameroon’s craters lakes in West Africa have been a focus of much study because they host species flocks (Trewavas et al., 1972; Stiassny et al., 1992; Schliewen et al., 1994; Schliewen & Klee, 2004; Neumann et al., 2011; Martin, 2012; Lamboj, 2014). In comparison, the African cichlid diversity outside of these regions appears to be extremely poor (Teugels & Thys van den Audenaerde, 2003). Only two species, the blackchin tilapia Sarotherodon melanotheron Rüppell 1852 and the Guinean tilapia Coptodon guineensis (Günther 1862), have been described so far in near-shore African marine environments, the latter being limited to Senegalese shores according to Stiassny et al. (2008). Some authors, however, reported the presence of C. guineensis in northern Moroccan localities: the Sebkhet Imlily, situated at c. 200 km from the border with Mauritania (Qninba et al., 2009), and 700 km to the North in rock pools (locally known as gueltas) along the Aabar wadi, tributary of Chbeyka wadi, Tan Tan province (south-western Morocco; Qninba et al., 2012). Similarly, while only one cichlid species is known in the Banc d’Arguin National Park (PNBA) in the north of Mauritania (Sevrin-Reyssac & Richer de Forges, 1985), Imraguen (the name given to local PNBA fishermen) distinguish two cichlids based on body colouration: ablack phenotype named Toumvertel kahla and a yellow phenotype named Toumvertel safra. Recent unpublished studies on cichlids targeted by fisheries at the PNBA also reported the presence of different phenotypes among the cichlids landed. All these independent observations call for further investigation to better describe the distribution range of cichlids in the Saharan area. Furthermore, because the taxonomic status of cichlids with widespread species has been controversial, such as for C. guineensis, the redbelly tilapia Coptodon zillii (Gervais 1848) and the redbreast tilapia Coptodon rendalli (Boulenger 1897) (for the list of synonymized species, see Eschmeyer, 2015), it is worthwhile to investigate the genetic status of these species further. In the past, genetic investigations of S. melanotheron demonstrated that this nominal species consisted of at least two species (and three sub-species) over its distribution from Congo to Mauritania (Falk et al., 1999, 2003). In this context, the first objective of this study was to investigate the phenotypic and genetic diversity of cichlids in the PNBA waters, by analysing their morphological and genetic variability, in order to clarify their taxonomic status. Secondly, the diversity of the tribe Coptodonini, and more specifically of some of their widespread species such as C. guineensis, C. rendalli or C. zillii, was evaluated by analysing several samples

of these species from remote locations over their distribution range. To recover the species diversity of this tribe and their phylogenetic relationships, a meta-analysis was performed including representative species of the different tribes and clades identified in the African cichlid phylogeny of Schwarzer et al. (2009) and Dunz & Schliewen (2013), and all cytochrome oxidase I (COI) and control region (CR) sequences labelled as C. guineensis, C. rendalli or C. zillii on GenBank.

MATERIALS AND METHODS

SAMPLING Samples were collected at two geographic scales: one local to investigate the genetic and morphometric diversity of cichlids in the PNBA and one continental to highlight the phylogenetic relationships among the PNBA cichlid lineages and those of West African cichlids. In the PNBA, fish sampling was carried out by Imraguen fishermen using fixed gillnets at three locations: R’Gueiba (19∘ 25′ 27⋅1′′ N; 16∘ 27′ 56⋅2′′ W), Teichott (19∘ 32′ 38⋅2′′ N; 16∘ 24′ 37⋅6′′ W) and Iwik (19∘ 53′ 08⋅5′′ N; 16∘ 17′ 43⋅0′′ W). Each of the 393 fish collected were measured, weighed and photographed. Soft dorsal fins were clipped and stored individually in tubes of 70% ethanol. The large-scale sampling targeted some widespread species belonging to Coptodonini (C. guineensis, C. rendalli and C. zillii) and Oreochromini (S. melanotheron). Samples were collected in various countries (Algeria, Egypt, Niger, Kenya, Malawi, Mauritania, Senegal, Sierra Leone, Cameroon, Côte d’Ivoire, Ghana and Gabon; for details, see Table I). When possible, voucher specimens (Table I) have been used as in-groups; in other cases, photographs of some specimens have been uploaded to the web site https://cichlidaebancarguinmauritania.shutterfly.com/.

MORPHOLOGICAL DATA AND ANALYSES The colour of each cichlid collected in the PNBA was examined immediately after landing, considering the presence of: (1) tilapian spot on the dorsal fin, (2) black stripes on flanks, (3) colour and spot pattern on the caudal fin and (4) colour of the lips and the body. Fishes storedon ice were then brought to the laboratory for morphometric analysis. A total of 21 morphometric measurements and six meristic counts were recorded from cichlids sampled in the PNBA. Mor- phometric measurements were taken, following Teugels & Thys van den Audenaerde (2003): standard length (LS), head length (HL), snout length (SnL), eye diameter (ED), interorbital width (IoW), preorbital bone length (PoL), width of the fifth ceratobranchial toothplate (WTPh), pharyngeal bone length (PhL), body depth (BD), caudal peduncle depth (CPD), caudal peduncle length (CPL), horizontal distance from front tip of snout to the articulation of first dorsal-fin ray (PdL), horizontal distance from front tip of snout to the articulation of first pectoral-fin ray (PpL), horizontal distance from front tip of snout to the articulation of first pelvic (ventral)-fin ray (PvL), pre-anal length (PaL), length of dorsal-fin base (LDFB), length of the longest dorsal-fin spine (LDFS), pectoral-fin length (PFL), pelvic (ventral)-fin length (VFL), length of the anal-fin base (AFL) and length of the third spine in the anal fin (L3SAF). Meristic counts are number of: dorsal-fin rays (NDR), dorsal-fin spines (NDS), anal-fin rays (NAR), anal-fin spines(NAS), scales along the lower lateral line (NSLL) and gill rakers on the first ceratobranchial (lower) gill arch (NGR). All measurements were recorded using a calliper and a ruler was used for LS. Because two phenotypes (A and B) were clearly identified from the colour pattern, morphometric and meristic differences between them were investigated through a principal component analysis (PCA) of ln-transformed morphometric data (21 measurements) using the statistical programme PAST 2.17c (Hammer et al., 2001). This PCA contains 119 specimens of type A and 274 specimens of type B. In this PCA, the first principal component (PC I) integrates the most size-related variation, whereas PC II, PC III and following components are theoretically size independent. A separate PCA was performed for the meristic dataset (six counts) alone;

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 4 N. G. KIDE ET AL. KJ938193 KJ938195 KJ938196 KJ938197 KJ925106 KJ925112KJ925113 KJ938204 KJ925114 KJ938205 KJ925115 KJ938206 KJ938207 GenBank accession number KJ938140KJ938141 KJ925110 KJ925111 KJ938202 KJ938203 KJ938129 KJ938128 KJ938183 S7 NDH2 CR COI KJ938089 KJ938147 KJ938079 KJ938134 KJ925105 KJ938194 KJ938080KJ938081 KJ938135KJ938082 KJ938136KJ938083 KJ925103 KJ938137KJ938084 KJ925104 KJ938138 KJ938192 KJ925108 KJ938139 KJ938198 KJ925109 KJ938199 KJ925107 KJ938200 KJ938201 KJ938078 KJ938133 KJ925102 KJ938191 GQ168095 GQ167781 GQ168090 GQ167776 R. Correira Sequences in bold are those produced in this study. available available No locality data No locality data duponti Alcolapia alcalica Chilochromis II-175II-176 CichlidaeII-22 type A CichlidaeII-3 type AII-6 Cichlidae PNBA, type Mauritania AII-60 PNBA, Mauritania CichlidaeII-63 type A N. G. Kidé CichlidaeIII-111 PNBA, type Mauritania A N. Cichlidae G. type Kidé AIII-227 Cichlidae PNBA, Mauritania type Cichlidae N. A type G.III-230 A Kidé PNBA, Mauritania Cichlidae typeIII-307 PNBA, A N. Mauritania G. Kidé Cichlidae typeIII-309 PNBA, A N. Mauritania PNBA, G. Mauritania Kidé Cichlidae N. type G.III-310 A Kidé PNBA, Mauritania Cichlidae N. type G.III-312 N. A Kidé G. PNBA, Kidé Mauritania Cichlidae typeIII-316 N. A G. PNBA, Kidé Mauritania Cichlidae typeIII-318 N. A G. PNBA, Kidé Mauritania Cichlidae typeT_3697 N. A G. PNBA, Kidé Mauritania Cichlidae type N. A G. PNBA, Kidé Cichlidae Mauritania type A N. G. PNBA, Kidé Mauritania N. G. PNBA, Kidé Mauritania N. PNBA, G. Mauritania Kidé N. G. Kidé C. Carneiro and E. J02 I-57II-17 Cichlidae type A Cichlidae type A PNBA, Mauritania PNBA, Mauritania N. G. Kidé N. G. Kidé Table I. Cichlid samples used in phylogenetic analyses, including sample code, species, location, collector nameCode and GenBank accession number. J08 Species Locality Collector Voucher I-10I-11 Cichlidae type B Cichlidae type B PNBA, Mauritania PNBA, Mauritania N. G. Kidé N. G. Kidé

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 A NEW COPTODON SPECIES IN WEST AFRICA? 5 KJ938185 KJ938186 KJ938188 KJ938189 KJ925118KJ925119 KJ938210 KJ925120 KJ938211 KJ925121 KJ938212 KJ938213 GenBank accession number KJ938144 KJ925122 KJ938214 S7 NDH2 CR COI KJ938090 KJ938148 KJ925069 KJ938230 KJ938075 KJ938130 KJ925099 KJ938184 KJ938076 KJ938131 KJ925100KJ938077 KJ938187 KJ938085 KJ938132KJ938086 KJ938142 KJ925101 KJ938143 KJ925116 KJ938190 KJ925117 KJ938208 KJ938209 GQ168136 GQ167822 GQ168143 GQ167829 GQ168117 GQ167803 GQ168139 GQ167825 ZSM40731 JX910809 JX910895 Table I. Continued A. Dunz available available Congo available available available Kisangani, DR No locality data No locality data No locality data No locality data No locality data zillii aff. aff. ‘Samou’ louka ‘Kisangani’ Coelotilapia joka Coptodon Coelotilapia joka Coptodon louka Coptodon Coelotilapia joka I-132 Cichlidae type B PNBA, Mauritania N. G. Kidé CodeI-130 Cichlidae type Species B PNBA, Mauritania N. G. Kidé Locality Collector Voucher I-19I-248I-249I-259 Cichlidae type B Cichlidae typeI-260 B Cichlidae typeIV-223 B PNBA, Cichlidae Mauritania typeIV-285 PNBA, B Mauritania Cichlidae typeIV-286 N. Cichlidae PNBA, B G. type Mauritania Kidé B N. G. Kidé Cichlidae PNBA, type Mauritania B N. G. Kidé Cichlidae PNBA, type Mauritania PNBA, B N. Mauritania G. Kidé PNBA, N. Mauritania G. N. Kidé G. Kidé PNBA, Mauritania N. G. Kidé N. G. Kidé IV-304-RIV-304-T Cichlidae type BIV-319 Cichlidae type BIV-58 PNBA, Mauritania114Tjo Cichlidae type B PNBA, Mauritania N. G. Cichlidae Kidé type B N. G. PNBA, Kidé Mauritania PNBA, N. Mauritania G. Kidé N. G. Kidé J67 ZSM-11 J33 J64 J72

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 6 N. G. KIDE ET AL. KJ925072 KJ938221 GenBank accession number S7 NDH2 CR COI KJ938057 KJ938103 KJ925065 KJ938056 KJ938102 KJ938235 KJ938092 KJ938150 KJ925070 KJ938224 KJ938055 KJ938101 KJ938234 KJ938054 KJ938100 KJ925064 KJ938233 GQ168135 GQ167821 GQ168115 GQ167801 TI142 JX910815 JX910883 Table I. Continued A. Lamboj TI102 JX910807 JX910885 A. Lamboj TI115 JX910811 JX910888 A. Lamboj TI117 JX910812 JX910886 I. SeidelA. Dunz ZSM40013A. Dunz JX910798 JX910877 GQ168135 GQ167821 Cameroon Cameroon Cameroon available Cameroon available Cameroon available available Aby, Cote d’Ivoire J.-F. Agnèse Lake Bermin, Aby, Cote d’Ivoire J.-F. Agnèse Aby, Cote d’Ivoire J.-F. Agnèse Lake Bermin, Aby, Cote d’Ivoire J.-F. Agnèse Lake Bermin, Ndian River, No locality data No locality data No locality data No locality data Lake Ejagham, guineensis bakossiorum guineensis guineensis bythobates guineensis camerunensis camerunensis Coptodon Coptodon Coptodon bemini Coptodon Coptodon Coptodon dageti Coptodon discolor Coptodon Coptodon Coptodon dageti Coptodon deckerti Coptodon Coptodon Code112Tba Species Locality Collector Voucher Cg-7447 116Tbe Cg-7446 117Tby Cg-7445 98Tcam J63 ZSM-02 120Tde J31 Cg-7444 ZSM-05

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 A NEW COPTODON SPECIES IN WEST AFRICA? 7 GenBank accession number KJ938106 KJ925067 KJ938159 KJ925069 KJ925069 S7 NDH2 CR COI KJ938059 KJ938105 KJ925066 KJ938158 KJ938058 KJ938104 KJ938236 KJ938060 KJ938108KJ938071 KJ925068 KJ938124KJ938072 KJ925094 KJ938179 KJ938125KJ938073 KJ925095 KJ938180 KJ938126KJ938074 KJ925096 KJ938181 KJ938127 KJ925097 KJ938182 KJ938049 KJ938095 KJ938153 KJ938050 KJ938096 KJ938154 GQ168151 GQ167837 Table I. Continued A. Pariselle A. Pariselle A. Pariselle A. Pariselle A. Pariselle A. Pariselle A. Pariselle A. Pariselle J.-F. Agnèse J.-F. Agnèse Senegal Senegal Senegal Senegal Makokou, Gabon Makokou, Gabon Makokou, Gabon Makokou, Gabon available of Accra, Ghana of Accra, Ghana Aby, Cote d’Ivoire J.-F. Agnèse Hann Bay, Dakar, Hann Bay, Dakar, Hann Bay, Dakar, Hann Bay, Dakar, Ivindo River, Ivindo River, Ivindo River, Ivindo River, No locality data Weija Lake, west Weija Lake, west guineensis guineensis guineensis guineensis guineensis guineensis guineensis guineensis guineensis guineensis guineensis guineensis Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon Coptodon CodeCg-7448 Species Locality Collector Voucher CgD-1 CgD-2 CgD-3 CgD-4 G59 G60 G92 G93 J84 Cg-5871 Cg-5872

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 8 N. G. KIDE ET AL. KJ925075 KJ938227 KJ925074 KJ938222 GenBank accession number S7 NDH2 CR COI KJ938069 KJ938118 KJ938172 KJ938068 KJ938117 KJ925089 KJ938171 KJ938067 KJ938116 KJ925088 KJ938170 KJ938093 KJ938151 KJ925071 KJ938226 KJ938065 KJ938113 KJ925086 KJ938167 KJ938053 KJ938099 KJ925063 KJ938157 KJ938052 KJ938098 KJ938156 KJ938051 KJ938097 KJ925062 KJ938155 Table I. Continued J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse A. Spreinat TI141 JX910814 JX910890 E. Swartz SAIAB85145 JX910803 JX910889 A. Dunz TI141 JX910814 JX910890 A. Dunz A. Dunz GQ168141 GQ167827 J.-F. Agnèse A. Dunz GQ168136 GQ167822 J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse Kenya Kenya Kenya Malawi falls, Angola Shire River, Malawi Cameroon Cameroon of Accra, Ghana available of Accra, Ghana of Accra, Ghana of Accra, Ghana Victoria Lake, Victoria Lake, Victoria Lake, Dja River, Lake Malawi, Dja River, Below Calema Weija Lake, west Lake Malawi near Weija Lake, west Weija Lake, west No locality data Weija Lake, west nyongana nyongana guineensis guineensis guineensis guineensis Coptodon rendalli Coptodon rendalli Coptodon rendalli Coptodon rendalli Coptodon rendalli Coptodon rendalli Coptodon Coptodon Coptodon louka Coptodon Coptodon Coptodon Coptodon Cz-6607 Cz-6606 Cz-6605 119Tre 104Tre ZSM-08 ZSM-07 J70 ZSM-03 Cz-5904 Cg-5879 Cg-5878 CodeCg-5877 Species Locality Collector Voucher

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 A NEW COPTODON SPECIES IN WEST AFRICA? 9 KJ938175 KJ938174 KJ925076 KJ938223 KJ925079 KJ938225 KJ925077 KJ938231 GenBank accession number KJ938120 KJ925090 KJ938176 S7 NDH2 CR COI KJ938070 KJ938119 KJ938173 KJ938094 KJ938152 KJ925078 KJ938228 KJ938088 KJ938146 KJ925126 KJ938220 KJ938087 KJ938145 KJ925125 KJ938219 ZSM38791 JX910820 JX910880 ZSM40782 JX910816 JX910887 Table I. Continued Schraml J.-F. Agnèse A. Dunz and E. J.-F. Agnèse A. DunzJ.-F. Agnèse ZSM38791 JX910820 JX910880 J.-F. Agnèse A. Dunz TI146 JX910817 JX910884 A. DunzA. Dunz GQ168139 GQ167825 GQ168118 GQ167804 A. Dunz Kenya Egypt d’Ivoire south-west of Alexandria, Egypt d’Ivoire Kenya Mamfe, Cameroon available available Cameroon available Cross River at No locality data Victoria Lake, Lake Bermin, Ourgla, AlgeriaLake Mariout, A. Pariselle Man, Cote Lake Mariout, Man, Cote Victoria Lake, No locality data Ourgla, Algeria A. Pariselle No locality data sp. sp. aff. ‘Samou’ ‘Cross’ louka snyderae Coptodon rendalli Coptodon rendalli Coptodon zillii Coptodon zillii Coptodon tholloni Coptodon zillii Coptodon zillii Coptodon zillii Coptodon Coptodon Coptodon Coptodon zillii Coptodon tholloni CodeCz-6608 Species Locality Collector Voucher Cz-6609 125Tzi Cz-7313 ZSM-06 Cz-7307 121Tsn ZSM-13 ZSM-04 J35 N2 ZSM-09 N1

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 10 N. G. KIDE ET AL. KJ925083KJ925084 KJ938164 KJ938165 GenBank accession number KJ938121 KJ925091KJ938122 KJ938177 KJ925092KJ938123 KJ938178 KJ925093 KJ938115 KJ925087 KJ938169 S7 NDH2 CR COI KJ938064 KJ938112KJ938066 KJ925085 KJ938114 KJ938166 KJ938168 KJ938061KJ938062 KJ938109KJ938063 KJ938110 KJ925080 KJ938111 KJ925081 KJ938161 KJ925082 KJ938162 KJ938163 GQ168091 GQ167777 Table I. Continued J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse J.-F. Agnèse A. Dunz ZSM35146 JX910804 JX910878 J. Gottwald PAA-443 JX910801 JX910893 of Accra, Ghana of Accra, Ghana of Accra, Ghana d’Ivoire d’Ivoire d’Ivoire at Kosti, Sudan available Mamfe, Cameroon Cross River at No locality data Ndjamena, NigerOrontes, SyriaWeija Lake, J.-F. west Agnèse Weija Lake, N. west Alwan et al.,Weija Lake, west ZSM40036 JX910790 JX910881 Man, Cote Man, Cote Man, Cote Ndjamena, NigerNdjamena, NigerNdjamena, J.-F. Niger Agnèse Ndjamena, J.-F. Niger Agnèse J.-F. Agnèse J.-F. Agnèse White Nile River ethelwynnae Etia nguti Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Coptodon zillii Gobiocichla 102Get Cz-5468 88Tzil Cz-5903 Cz-5905 Cz-5906 Cz-7319 Cz-7322 Cz-5462 Cz-5463 Cz-5464 Cz-5465 105Tzi CodeCz-7316 Species Locality Collector Voucher J03

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 A NEW COPTODON SPECIES IN WEST AFRICA? 11 KJ925098 KJ938229 GenBank accession number S7 NDH2 CR COI TI150 JX910819 JX910871 TI149 JX910818 JX910870 SAIAB84681 JX910802 JX910874 and D. Neto Table I. Continued Cameroon Cameroon Freetown, Sierra Leone A. Dunz GQ168111 AF317251 No locality data available GQ168111 GQ167797 No locality data available GQ168144 GQ167830 No locality data available GQ168119 GQ167805 Lake Barombi Mbo, No locality data available GQ168094 GQ167780 No locality data available GQ168096 GQ167782 No locality data available GQ168133 GQ167819 Congo River, DR Congo D. Neumann ZSM37722 JX910793 JX910869 Lucalla River, Angola E. Swartz, R. Bills No locality data available ZSM39713 JX910800 JX910876 Lake Barombi Mbo, No locality data available GQ168101 GQ167787 No locality data available GQ168125 GQ167811 buttikoferi buttikoferi hormuzensis andersonii niloticus tanganicae caudomarginatus galilaeus lohbergeri nigripinnis Heterotilapia Heterotilapia Iranocichla Konia eisentrauti Oreochromis Oreochromis Oreochromis Pelmatolapia cabrae Pelmatolapia mariae Sarotherodon Sarotherodon Sarotherodon Sarotherodon mvogoi Sarotherodon J27 CodeZSM-10 Species Locality Collector Voucher J74 124Kei J38 J07 J10 103Tca 101Tma J61 92Sgal 123Slo J49 J15

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 12 N. G. KIDE ET AL. KJ938218 KJ938216 KJ925123 KJ938215 KJ925124 KJ938217 GenBank accession number S7 NDH2 CR COI KJ938091 KJ938149 KJ925073 KJ938232 ZSM36123 JX910794 JX910894 G. Baffur Entwi A. Dunz Table I. Continued ; PNBA, Banc d’Arguin National Park; TI and ZSM, Zoologisches Staatssammlung München; SAIAB, South African Abono, Ghana Guiers Lake, SenegalGuiers Lake, Senegal J.-D. Durand J.-D. Durand Guiers Lake, Senegal J.-D. Durand Guiers Lake, Senegal J.-D. Durand Sanaga River, Cameroon A. Lamboj ZSM38845 JX910805 JX910868 No locality data availableNo locality data available GQ168103 GQ167789 GQ168130 GQ167816 No locality data available GQ168110 GQ167796 Anum River, GhanaRD Congo D. Neumann, U. Schliewen ZSM40760 JX910806 JX910826 Lake Bosumtwi, village No locality data available GQ168142 GQ167828 No locality data available GQ168114 GQ167800 No locality data available GQ168127 GQ167813 control region , CR ; melanotheron melanotheron melanotheron melanotheron sanagaensis Steatocranus bleheri Steatocranus glaber Sarotherodon Sarotherodon Sarotherodon Sarotherodon Sarotherodon Stomatepia mariae Tilapia busumana Tilapia brevimanus Tilapia ruweti Tilapia sparrmanii Tilapia pra Tristramella simonis cytochrome oxidase I , J18 J55 Lgu4-E Lgu7-E Lgu10-E Lgu1-E Code108Ssa Species Locality Collector Voucher COI J25 Institute for Aquatic Biodiversity. ZSM-12 J71 111Tru J30 ZSM36123 J51

meristics are size independent and thus PC I is the most informative component. Scores of both PCAs were combined in a single bivariate plot for most informative visualization.

MOLECULAR DATA AND ANALYSES Phylogenetic relationships among cichlids sampled in PNBA (Mauritania), Hann Bay (Dakar, Senegal) and others belonging to Oreochromini and Coptodonini (Dunz & Schliewen, 2013) (in-group) were estimated using sequence polymorphisms of a mitochondrial (ND2)anda nuclear (S7 ribosomal protein first intron) gene. ND2 and S7 sequences published by Schwarzer et al. (2009) and Dunz & Schliewen (2013) were downloaded from GenBank and used as reference species because these contain sequences of voucher specimens stored in museums used for a revision of cichlid systematics from Africa (Dunz & Schliewen, 2013). To investigate the phylogeographic structure and provide insight into the intraspecific genetic diversity of widespread species of Coptodon Gervais 1853 (C. guineensis, C. rendalli and C. zillii)aswellasS. melanotheron,theCOI portion and the mitochondrial CR fragment were also sequenced. These two additional mitochondrial sequences were chosen because: (1) the COI sequence is the marker used for DNA barcoding in the BOLD programme, so data are available on GenBank and can be used to increase the number of locations analysed for a species; (2) the CR is known for its high level of polymorphism and was used by Falk et al. (2003) to investigate the phylogeographic structure of S. melanotheron and Sarotherodon nigripinnis (Guichenot 1861) and (3) COI and CR sequences available in GenBank of species of Coptodon and S. melanotheron were added to the dataset (Table SI, Supporting Information). Only COI sequences of barcoding studies conducted in Africa were downloaded while most of CR sequences added to the present analyses belong to the study of Falk et al. (2003). Genomic DNA was extracted from fin clips using DNeasy Blood & Tissue kit (QIAGEN; www.qiagen.com) following the standard protocol provided by the manufacturer. Amplifica- tions were performed in 25 μl volumes containing 1 μl of genomic DNA, 3 mM of forward and reverse primers, 12⋅5 μl of FastStart PCR Master mix (Roche; https://lifesciences.roche.com/), 0⋅5 μl of 2% bovine serum albumin (Euromedex; www.euromedex.com) and water. Amplifica- tion of all fragments were carried out in 30–35 cycles according to the following temperature profile: 3 min at 92∘ C (initial denaturation), 45 s at 92∘ C, 45 s at 52∘ C (for ND2 and COI) or 56∘ C (for CR), 60 s at 72∘ C and finally 5 min at 72∘ C. Specifically for the S7 sequence amplification, a touch down PCR was adapted from the previous PCR conditions and where annealing temperature decreases 0⋅5∘ C each cycle from 62 to 58∘ C, totaling 35 cycles. Primers used to amplify the different gene portion were ND2Met/ND2Trp (Kocher et al., 1995) for ND2, S7RPEX1F50/S7RPEX2R50 for S7 (Chow & Hazama, 1998), FishF1 + FishF2/FishR1 for COI (Ward et al., 2005) and L-Smel/H-Smel for CR (Falk et al., 2003). Portions of the nuclear intronic (S7) and mitochondrial CR sequences were aligned using Muscle (Edgar, 2004) as implemented in MEGA 5.2.2 (Tamura et al., 2011) while COI and ND2 gene sequences were aligned visually using the alignment editor provided by MEGA 5.2.2. Phylogenetic analyses were conducted applying the maximum likelihood (ML) approach implemented in MEGA 5.2.2. Best-fit models of sequence evolution for each dataset were estimated using the option «Best DNA model» implemented in MEGA 5.2.2 (www.megasoftware.net). A first phylogenetic analysis was performed on ND2 and S7 sequences to highlight phylogenetic relationships of the samples with those analysed by Schwarzer et al. (2009) and Dunz & Schliewen (2013). To evaluate phylogenetic information recovered by each gene, single gene trees were built. A second phylogenetic analysis was performed on COI and CR sequences to investigate the genetic diversity of C. guineensis, C. rendalli, C. zillii and S. melanotheron. The Hasegawa Kishino Yano (HKY) model, considering non-uniformity of evolutionary rates among sites modelled using a discrete gamma distribution (+G), was used for ND2 + S7 and CR datasets [Bayesian information criterion (BIC) = 19594⋅73 and 7965⋅32, respectively]; Kimura two-parameter (K2P) model, +GforCOI (BIC = 8014⋅0) and invariant mutation rates among sites (+I) for S7 (BIC = 5427⋅706). Node support was tested using the bootstrap procedure (Felsenstein, 1985), based on 200 pseudo-replicates. All phylogenetic trees were rooted using the out-groups Heterochromis multidens (Pellegrin 1900) and Etia nguti Schliewen & Stiassny 2003 following Dunz & Schliewen (2013).

(a) (c)

(b) (d)

Fig. 1. Cichlidae collected in the Banc d’Arguin National Park, Mauritania: (a, b) phenotype A with colour variation and (c, d) phenotype B with its colour variation. Scale bar (white + grey) represents 2 cm.

RESULTS

PHENOTYPES, MORPHOMETRY AND MERISTICS Different phenotypes were observed in the cichlids sampled in PNBA, based upon the general colour of body and fins. Although there was wide morphological variation, all individuals could be classified into two main phenotypes. Cichlids classified as phenotype A were characterized by: presence of a tilapian spot on dorsal fin, caudal fin spotted, greyish on its upper parts and yellowish in lower parts; the belly canpresent pinkish to yellowish colour while the back is brownish; large vertical bars present on sides [Fig. 1(a)]. Cichlids classified as phenotype B were characterized by: presence of a white lip (lower) and absence of spots (including the tilapian spot) on dorsal and caudal fins [Fig. 1(c)]. Large colour variation was observed within the two phenotypes, but the colour of phenotype A was usually lighter and side bands were less marked [Fig. 1(b)], while specimens of phenotype B were usually totally black [Fig. 1(d)]. Additional photographs of some specimens used in this study are available at the web- site: https://cichlidaebancarguinmauritania.shutterfly.com/. A PCA confirms the differences observed between specimens classified as phenotype A or B (Fig. 2). Differences between the two phenotypes are mainly noticeable on the PCA I meristic axis that accounts for 81⋅43% of the total variance while the PCA II morphometric axis accounts for 10⋅75% (Fig. 2). When compared with B, specimens classified as phenotype A have shorter PFL, HL, PpL and PhL. Additionally, theNDR is higher in phenotype A v. B, while NGR is lower in phenotype A v. B (Table II).

PHYLOGENETIC RELATIONSHIPS All sequences obtained in this study have been deposited on GenBank (Table I). Fragment size ranges between 355 and 371 for the CR, 469 and 492 bp for S7, 632 and 648 bp for COI and 972 bp for ND2. In all reconstructed phylogenetic trees, cichlids collected in PNBA belong to two divergent clades. These two clusters are com- posed of the same individuals presenting the same phenotype, independent of the gene

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Fig. 2. Bivariate plot of principal component II (PC II; morphometrics) and PC I (meristics); 95% c.i. visualized as ellipses. ,typeA(n = 119); , type B (n = 340). Total number of specimens in the plot (n = 459). Labelled specimens 1–11 refer to the type B. sequences used for the phylogenetic reconstruction, with one exception: individual I-57 that appears in clade B in the nuclear S7 tree [Fig. 3(a)] and in the clade A in mitochondrial trees [Figs 3(b) and 4]. The S7 phylogenetic tree [Fig. 3(a)] presents a lower resolution than the ND2 mitochondrial tree [Fig. 3(b)] or the multi-gene phylogenetic tree published by Dunz & Schliewen (2013), but the phylogenetic relationships highlighted by the S7 tree are largely concordant, and even more highly supported than the ND2 tree for deep divergences. Thus, in the S7 tree [Fig. 3(a)] the Etini tribe is the most divergent haplotilapiine followed by the Oreochromini tribe, as recovered in the multi-gene tree (Dunz & Schliewen, 2013). The Oreochromini and Coptodonini tribes are also monophyletic in the S7 tree, whereas the node basal to all species of the Coptodonini tribe is not supported in the ND2 tree due to the placement of the two specimens of Coptodon tholloni (Sauvage 1884) [CIII; Fig. 3(b)]. For recent divergences, the two Oreochromini clades SI and SII are not supported in the S7 tree, unlike on the ND2 tree [Fig. 3(a), (b)]; but the three Coptodonini clades (CI, CII and CIII) are well supported in the two single gene trees. Species composition of these last three clades is identical in both trees, with one exception. While morphotype A is grouped with C. guineensis sampled in Senegal and belongs to the CI clade in the S7 tree, this group of sequences belongs to the CII clade in the ND2 tree [Fig. 3(a), (b)]. Lastly, in both trees, C. guineensis is polyphyletic or paraphyletic. The phylogenetic tree built from the concatenation of both ND2 and S7 sequences (I-57 has been removed) highlights a close relationship of individuals classified as phenotype A with C. guineensis collected in Senegal, and belong to a subclade including Coptodon sp. aff. louka and Coptodon camerunensis (Lönnberg 1903), within the clade CII of the Coptodonini tribe [Fig. 3(c)]. Individuals classified as phenotype B present a close relationship with Sarotherodon nigripinnis (Guichenot 1861), all included in the

Table II. Morphometric variability and meristic counts (in italics) recorded on Mauritanian cichlids presenting the phenotypes A (n = 97) and B (n = 296)

Phenotype A Phenotype B Mean ± s.d. Mean ± s.d. Morphometric %HL 23⋅6 ± 4⋅028⋅4 ± 2⋅8 variability %SnL 11⋅6 ± 2⋅512⋅5 ± 1⋅5 %ED 4⋅8 ± 0⋅65⋅2 ± 0⋅6 %IoW 8⋅6 ± 1⋅29⋅5 ± 1⋅0 %PoL 7⋅1 ± 1⋅18⋅6 ± 1⋅2 %WTPh 6⋅2 ± 0⋅86⋅1 ± 0⋅8 %PhL 7⋅7 ± 1⋅010⋅2 ± 1⋅2 %BD 31⋅4 ± 3⋅233⋅3 ± 2⋅8 %CPD 14⋅1 ± 1⋅714⋅5 ± 1⋅3 %CPL 9⋅6 ± 1⋅310⋅1 ± 1⋅4 %PdL 23⋅0 ± 3⋅124⋅8 ± 3⋅2 %PpL 22⋅9 ± 2⋅626⋅9 ± 2⋅6 %PvL 28⋅7 ± 3⋅231⋅5 ± 3⋅5 %PaL 55⋅9 ± 5⋅758⋅8 ± 6⋅0 %LDFB 45⋅3 ± 4⋅747⋅1 ± 3⋅7 %LDFS 9⋅3 ± 2⋅18⋅8 ± 1⋅3 %PFL 25⋅1 ± 3⋅634⋅7 ± 4⋅0 %VFL 23⋅4 ± 3⋅720⋅9 ± 2⋅9 %AFL 13⋅1 ± 1⋅613⋅7 ± 1⋅3 %L3SAF 7⋅6 ± 1⋅28⋅4 ± 1⋅0 Most Minimum– Most Minimum– frequent maximum frequent maximum Meristic counts NDR 14 12–15 12 11–14 NDS 15 14–16 16 15–17 NAR 10 9–11 10 8–11 NAS 3333 NSLL 29 27–32 29 27–32 NGR 18 16–20 24 20–26

%, Measure corrected by the standard length (LS). HL, head length; SnL, snout length; ED, eye diameter; IoW, interorbital width; PoL, preorbital bone length; WTPh, width of the fifth ceratobranchial toothplate; PhL, pharyngeal bone length; BD, body depth; CPD, caudal peduncle depth; CPL, caudal peduncle length; PdL, horizontal distance from front tip of snout to the articulation of first dorsal-fin ray; PpL, horizontal distance from front tip of snout to the articulation of first pectoral-fin ray; PvL, horizontal distance from front tip of snout to the articulation of first pelvic (ventral)-fin ray; PaL, pre-anal length; LDFB, length of dorsal-fin base; LDFS, length of the longest dorsal-fin spine; PFL, pectoral-fin length; VFL,pelvic (ventral)-fin length; AFL, length of the anal-fin base; L3SAF, length of the third spine in the anal fin;NDR, dorsal-fin rays; NDS, dorsal-fin spines; NAR, anal-fin rays; NAS, anal-fin spines; NSLL, scales alongthe lower lateral line; NGR, gill rakers on the first ceratobranchial (lower) gill arch. clade SI inside the Oreochromini tribe. Samples of C. zillii analysed in this study are all grouped with other C. zillii analysed by Dunz & Schliewen (2013), just as the current samples of C. rendalli are also grouped with C. rendalli analysed by Schwarzer et al. (2009) and Dunz & Schliewen (2013). In contrast, the current samples of C. guineensis, identified in the field by morphological characteristics, are included in three different

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subclades within two clades of the Coptodonini tribe. The samples of C. guineensis from Ghana are closely related to C. guineensis analysed by Dunz & Schliewen (2013), Coptodon discolor (Günther 1903) and Coptodon dageti (Thys van den Audenaerde 1971), all included in the clade CII [Fig. 3(c)]. Coptodon guineensis sampled in Senegal also belongs to this clade, but to a different subclade. Coptodon guineensis sampled in Gabon belongs to the clade CI in a subclade that includes C. rendalli, C. zillii, C. sp. aff. zillii ‘Kisangani’, Coptodon nyongana (Thys van den Aude- naerde 1971), Coptodon sp. ‘Cross’, Coptodon deckerti (Thys van den Audenaerde 1967), Coptodon bemini (Thys van den Audenaerde 1972), Coptodon bythobates (Stiassny, Schliewen & Dominey 1992), Coptodon snyderae (Stiassny, Schliewen & Dominey 1992) and Coptodon bakossiorum (Stiassny, Schliewen & Dominey 1992) [Fig. 3(c)]. Phylogenetic relationships inferred from the COI sequence polymorphisms confirm the presence of two species in the Mauritanian cichlid samples, and that they belong to two different genera and tribes [Fig. 4(a)]. This COI tree confirms the monophyly of C. zillii that consists of two subclades: one group including all individuals sampled exclusively in Côte d’Ivoire [subclade Cz-II in Figs 4(a) and 5(b)] and the second group including all other C. zillii sampled over a large geographic range: from north Africa and the Levant in the north, to Niger, Ghana and Nigeria to the south [subclade Cz-I in Figs 4(a) and 5(b)]. The polyphyly of C. guineensis is confirmed and consists of four lineages in this COI tree, as sequences of C. guineensis from Nigeria uploaded from GenBank were grouped with Coptodon sp. ‘Cross’ [subclade Cg-III in Figs 4(a) and 5(a)]. Lastly, the phylogenetic relationships inferred from CR also support previous observations; fine intraspecific relationships are highlighted, especially in S. melanotheron where two subclades are retrieved. One grouped all individuals sampled in Ghana and Côte d’Ivoire [subclade Sm-II in Figs 4(b) and 5(d)] and the other one consisted of individuals sampled in Liberia, Sierra Leone, Senegal and Mauritania [subclade Sm-I in Figs 4(b) and 5(d)].

DISCUSSION

SPECIES DIVERSITY AND POLYCHROMATISM OF PNBA CICHLIDS Both morphometric and genetic analyses clearly demonstrate the presence of two cichlid species in PNBA, Mauritania. To date, only S. melanotheron was recorded in

Fig. 3. Phylogenetic relationships among Mauritanian cichlids (types A and B) and other haplotilapiine species belonging to clade and tribes (with the exception of East African cichlids radiation) depicted in Dunz & Schliewen (2013). Maximum likelihood (ML) analyses are based on polymorphisms of (a) S7 sequences, (b) ND2 and (c) the concatenation of S7 and ND2 sequences. Branch length is proportional to the number of substitutions under (a) Kimura two-parameter (K2P) + I, (b) Hasegawa Kishino Yano (HKY) + G + Iand (c) HKY + G model. Numbers on the branches are ML bootstrap values (in %, from 200 replicates), with those below 70% not shown. Scale bar: 0⋅1 inferred nucleotide substitution/site under (HKY + G) model. , point to an individual that presents different placement in phylogenetic reconstructions.

JF510525 - Coptodon zillii, Nigeria Cz-5465 - Coptodon zillii, Niger Cz-5464 - Coptodon zillii, Niger HM882906 - Coptodon zillii, Nigeria Cz-5462 - Coptodon zillii, Niger (a) HM882904 - Coptodon zillii, Nigeria (b) ZSM-06 - Coptodon zillii, Egypt HM882894 - Coptodon zillii, Nigeria Cz-5468 - Coptodon zillii, Niger HM882888 - Coptodon zillii, Nigeria 94 Cz-5463 - Coptodon zillii, Niger FJ348135 - Coptodon zillii, Israel EU163719 - Coptodon zillii, Israel FJ348134 - Coptodon zillii, Israel FJ613479 - Coptodon zillii, Israel Cz-I FJ348133 - Coptodon zillii, Israel Cz-5903 - Coptodon zillii, Ghana FJ348132 - Coptodon zillii, Israel 90 Cz-5906 - Coptodon zillii, Ghana Cz-5906 - Coptodon zillii, Ghana 98 N2 - Coptodon zillii, Algeria Cz-5905 - Coptodon zillii, Ghana N1 - Coptodon zillii, Algeria Cz-5903 - Coptodon zillii, Ghana Cz-7313 - Coptodon zillii, Cote d’Ivoire Cz-5468 - Coptodon zillii, Niger Cz-7316 - Coptodon zillii, Cote d’Ivoire Cz-5465 - Coptodon zillii, Niger 98 Cz-7322 - Coptodon zillii, Cote d’Ivoire Cz-II Cz-7319 - Coptodon zillii, Cote d’Ivoire Cz-5464 - Coptodon zillii, Niger Cz-I G93 - Coptodon guineensis, Gabon Cz-5463 - Coptodon zillii, Niger 99 G59 - Coptodon guineensis, Gabon Cz-5462 - Coptodon zillii, Niger G92 - Coptodon guineensis, Gabon Cg-IV N1 - Coptodon zillii, Algeria 75 G60 - Coptodon guineensis, Gabon N2 - Coptodon zillii, Algeria ZSM-07 - Coptodon nyongana, Cameroon KJ552862 - Coptodon zillii, Algeria 99 ZSM-08 - Coptodon rendalli, Malawi KJ552984 - Coptodon zillii, Syria AF296503 - Coptodon rendalli, Zimbabwe 76 KJ553213 - Coptodon zillii, Jordan 80 71 Cz-6606 - Coptodon rendalli, Kenya ZSM-06 - Coptodon zillii, Egypt 90 Cz-6605 - Coptodon rendalli, Kenya FJ348136 - Coptodon zillii, Israel ZSM-13 - Coptodon sp. “Cross”, Cameroon Cg-III KJ553249 - Coptodon zillii, Morocco ZSM-09 - Coptodon tholloni KJ553295 - Coptodon zillii, Morocco ZSM-02 - Coptodon dageti 98 Cz-5904 - Coptodon guineensis, Ghana KJ553279 - Coptodon zillii, Syria 73 99 Cg-7444 - Coptodon guineensis, Cote d’Ivoire HM882889 - Coptodon zillii, Nigeria Cg-5879 - Coptodon guineensis, Ghana Cg-I 84 JF510524 - Coptodon zillii, Syria 94 Cg-5877 - Coptodon guineensis, Ghana 76 Cz-7307 - Coptodon zillii, Cote d’Ivoire Cg-7447 - Coptodon guineensis,Cote d’Ivoire Cz-7313 - Coptodon zillii, Cote d’Ivoire ZSM-03 Coptodon louka Cz-7316 - Coptodon zillii, Cote d’Ivoire Cz-II ZSM-05 - Coptodon camerunensis Cz-7319 - Coptodon zillii, Cote d’Ivoire ZSM04 - Coptodon sp. aff louka “Samou” 74 G60 - Coptodon guineensis, Gabon 88 CgD-1 - Coptodon guineensis, Sénégal 99 G92 - Coptodon guineensis, Gabon CgD-4 - Coptodon guineensis, Sénégal G59 - Coptodon guineensis, Gabon Cg-IV CgD-2 - Coptodon guineensis, Sénégal G93 - Coptodon guineensis, Gabon 99 I-57 - Mauritania 77 ZSM-07 - Coptodon nyongana, Cameroon II-175 - Mauritania 98 II 60 - Mauritania ZSM-08 - Coptodon rendalli, Malawi II 63 - Mauritania 99 Cz-6605 - Coptodon rendalli, Kenya II-6 - Mauritania Cz-6606 - Coptodon rendalli, Kenya III-111 - Mauritania Cg-II 99 Cz-6607 - Coptodon rendalli, Kenya III-227 - Mauritania Cz-6608 - Coptodon rendalli, Kenya 85 III 230 - Mauritania Cz-6609 - Coptodon rendalli, Kenya III-307 - Mauritania A 85 ZSM-13 - Coptodon sp. “Cross”, Cameroon III-309 - Mauritania HM882911 - Coptodon guineensis, Nigeria III-310 - Mauritania 99 III-312 - Mauritania HM882907 - Coptodon guineensis, Nigeria III-318 - Mauritania HM882908 - Coptodon guineensis, Nigeria Cg-III III-316 - Mauritania 85 HM882895 - Coptodon guineensis, Nigeria ZSM-10 - Heterotilapia buttikoferi 70 Coptodon guineensis, 75 HM882893 - Nigeria 80 ZSM-11 - Coelotilapia joka HM418203 - Coptodon tholloni, Congo 85 AF485086 - Sarotherodon galileus, Ghana 98 79 HM418204 - Coptodon tholloni, Congo 98 AF485084 - Sarotherodon galileus, Ghana 99 ZSM-09 - Coptodon tholloni AF485085 - Sarotherodon galileus, Ghana HM418205 - Coptodon tholloni, Congo AF328852 - Sarotherodon galilaeus, Kenya AF484104 - Sarotherodon nigripinnis, Congo ZSM-10 - Heterotilapia buttikoferi 72 AF484103 - Sarotherodon nigripinnis, Congo ZSM-02 - Coptodon dageti 86 AF484105 - Sarotherodon nigripinnis, Congo Cg-7446 - Coptodon guineensis, Cote d’Ivoire AF484101 - Sarotherodon nigripinnis, Congo Cg-7448 - Coptodon guineensis, Cote d’Ivoire AF484102 - Sarotherodon m. nigripinnis, Congo Cg-7445 - Coptodon guineensis, Cote d’Ivoire 86 AF484106 - Sarotherodon nigripinnis, Congo 85 Cg-7444 - Coptodon guineensis, Cote d’Ivoire AF484099 - Sarotherodon nigripinnis, Gabon Cz-5904 - Coptodon guineensis, Ghana AF484098 - Sarotherodon nigripinnis, Gabon Cg-5879 - Coptodon guineensis, Ghana Cg-I 75 AF484100 - Sarotherodon nigripinnis, Gabon 99 AF484097 - Sarotherodon nigripinnis, Gabon Cg-5872 - Coptodon guineensis, Ghana 88 AF484737 - Sarotherodon melanotheron, Cote d’Ivoire Coptodon guineensis, 91 Cg-5871 - Ghana AF484720 - Sarotherodon melanotheron, Cote d’Ivoire Cg-5877 - Coptodon guineensis, Ghana AF484741 - Sarotherodon melanotheron, Ghana 97 Cg-5878 - Coptodon guineensis, Ghana AF484738 - Sarotherodon melanotheron, Ghana ZSM-03 - Coptodon louka AF484718 - Sarotherodon melanotheron, Cote d’Ivoire 94 ZSM-05 - Coptodon camerunensis AF484742 - Sarotherodon melanotheron, Ghana ZSM-04 - Coptodon sp. aff. louka “Samou” AF484740 - Sarotherodon melanotheron, Ghana CgD-2 - Coptodon guineensis, Senegal AF484739 - Sarotherodon melanotheron, Ghana AF484725 - Sarotherodon melanotheron, Cote d’Ivoire 99 CgD-1 - Coptodon guineensis, Senegal AF484732 - Sarotherodon melanotheron, Cote d’Ivoire CgD-3 - Coptodon guineensis, Senegal AF484743 - Sarotherodon melanotheron, Ghana II-63 - Mauritania 81 AF484717 - Sarotherodon melanotheron, Cote d’Ivoire I-57 - Mauritania AF484719 - Sarotherodon melanotheron, Cote d’Ivoire II-17 - Mauritania AF484724 - Sarotherodon melanotheron, Cote d’Ivoire II-175 - Mauritania 98 AF484734 - Sarotherodon melanotheron, Cote d’Ivoire II-176 - Mauritania AF484721 - Sarotherodon melanotheron, Cote d’Ivoire Sm-II II-22 - Mauritania AF484733 - Sarotherodon melanotheron, Cote d’Ivoire II-3 - Mauritania AF484731 - Sarotherodon melanotheron, Cote d’Ivoire AF484728 - Sarotherodon melanotheron, Cote d’Ivoire II 60 - Mauritania 99 AF484735 - Sarotherodon melanotheron, Cote d’Ivoire III-111 - Mauritania A Cg-II AF484730 - Sarotherodon melanotheron, Cote d’Ivoire III-227 - Mauritania 80 AF484726 - Sarotherodon melanotheron, Cote d’Ivoire III-230 - Mauritania AF484729 - Sarotherodon melanotheron, Cote d’Ivoire III-307 - Mauritania AF484727 - Sarotherodon melanotheron, Cote d’Ivoire III-309 - Mauritania AF484723 - Sarotherodon melanotheron, Cote d’Ivoire III-310 - Mauritania AF484736 - Sarotherodon melanotheron, Cote d’Ivoire III-312 - Mauritania AF484722 - Sarotherodon melanotheron, Cote d’Ivoire 84 AF484705 - Sarotherodon melanotheron, Sierra Leone III-316 - Mauritania AF484704 - Sarotherodon melanotheron, Sierra Leone III-318 - Mauritania AF484716 - Sarotherodon melanotheron, Liberia ZSM-11 - Coelotilapia joka AF484708 - Sarotherodon melanotheron, Liberia FJ348128 - Sarotherodon galilaeus, Israel AF484709 - Sarotherodon melanotheron, Liberia FJ348127 - Sarotherodon galilaeus, Israel AF484712 - Sarotherodon melanotheron, Liberia 93 FJ348129 - Sarotherodon galilaeus, Israel AF484715 - Sarotherodon melanotheron, Liberia 86 FJ348130 - Sarotherodon galilaeus, Israel AF484714 - Sarotherodon melanotheron, Liberia FJ348131 - Sarotherodon galilaeus, Israel AF484713 - Sarotherodon melanotheron, Liberia HM882897 - Sarotherodon galilaeus, Nigeria AF484707 - Sarotherodon melanotheron, Sierra Leone 72 AF484711 - Sarotherodon melanotheron, Liberia I-248 - Mauritania 86 AF484710 - Sarotherodon melanotheron, Liberia IV-286 - Mauritania AF484706 - Sarotherodon melanotheron, Sierra Leone I-11 - Mauritania AF484700 - Sarotherodon melanotheron, Senegal Sm-I 99 I-130 - Mauritania AF484702 - Sarotherodon melanotheron, Senegal I-132 - Mauritania LguE10 - Sarotherodon melanotheron, Senegal I-19 - Mauritania AF484699 - Sarotherodon melanotheron, Senegal Sarotherodon melanotheron I-249 - Mauritania AF484697 - , Senegal AF484698 - Sarotherodon melanotheron, Senegal I-259 - Mauritania B AF484701- Sarotherodon melanotheron, Senegal I-260 - Mauritania 99 AF484703- Sarotherodon melanotheron, Senegal IV-223 - Mauritania Sm-I Lgu4-E - Sarotherodon melanotheron, Senegal IV-285 - Mauritania IV-304 - Mauritania IV-304 - Mauritania I-130 - Mauritania IV-304-T - Mauritania I-248 - Mauritania I-260 - Mauritania IV-319 - Mauritania IV-223 - Mauritania IV-58 - Mauritania IV-286 - Mauritania Lgu1-E - Sarotherodon melanotheron, Senegal IV-304 - Mauritania B Lgu10-E - Sarotherodon melanotheron, Senegal IV-58 - Mauritania Lgu4-E - Sarotherodon melanotheron, Senegal IV-319 - Mauritania 0·02 0·05 Lgu7-E - Sarotherodon melanotheron, Senegal IV-285 - Mauritania

Fig. 4. Phylogenetic relationships among Mauritanian cichlids (types A and B) and other species belonging to Coptodonini, Oreochromini, Heterotilapiini and Coelotilapini tribes as depicted in Dunz & Schliewen (2013). Maximum likelihood (ML) analyses are based on polymorphisms of (a) cytochrome oxidase I portion (COI)and(b)control region (CR). Branch length is proportional to the number of substitutions under (a) Kimura two-parameter (K2P) + G and (b) Hasegawa Kishino Yano (HKY) + G models. Numbers on the branches are ML bootstrap values (in %, from 200 replicates), with those below 70% not shown. Scale bar: 0⋅1 inferred nucleotide substitution/site under (K2P + G or HKY + G + I) model. (a) In the COI tree, Coptodon guineensis and Coptodon zillii subclades are highlighted ( ). (b) In the CR tree, Sarotherodon melanotheron subclades are highlighted ( ).

40 (a) (b)

−20 40 (c) (d)

−20 −30 −15 01530−30 −15 01530

Fig. 5. Distribution of clades of (a) Coptodon guineensis,(b)Coptodon zillii,(c)Coptodon rendalli and (d) Sarotherodon melanotheron sensu lato highlighted in phylogenetic analyses based on cytochrome oxidase I (COI)andcontrol region (CR) [Fig. 4(a), (b)]. , Cg-II; , Cg-I; , Cg-III; , Cg-IV; , Cz-I; , Cz-II; , C. rendalli; , Sm-I; , Sm-II; , Sarotherodon nigripinnis. Shaded border areas correspond to distribution range of these species or species complex. this area, which exhibits the phenotype B, as classified by this study. All morphometric variables, meristic counts and phylogenetic relationships of phenotype B cichlids are in agreement with the description of S. melanotheron. This species is abundant in estuarine environments over its distribution range from Cameroun to Mauritania (Teugels & Thys van den Audenaerde, 2003). In contrast, the report of a second species is unexpected. Meristic and morphometric parameters recorded on individuals exhibiting phenotype A match with the description of a species belonging to Coptodon. Its presence in a marine environment and its close phylogenetic relationships with C. guineensis collected in Hann Bay (Dakar, Senegal), however, suggest that this species is C. guineensis (but see below). This species is widely distributed in West Africa,

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.12899 A NEW COPTODON SPECIES IN WEST AFRICA? 21 from Angola to Senegal (Teugels & Thys van den Audenaerde, 2003). Its known northern distribution limit corresponds to the Senegal River estuary, at the border between Mauritania and Senegal. Populations attributed to this species, however, have recently been reported at more northern localities: in the Sebkhet Imlily (Morocco), situated at c. 200 km from the border with Mauritania in salt waters (Qninba et al., 2009), and in rock pools (locally known as gueltas) along the Aabar wadi, tributary of Chbeyka wadi, Tan Tan province (south-western Morocco; Qninba et al., 2012). Therefore, the presence of C. guineensis in PNBA is not completely surprising, even if previous records were absent and despite the many marked morphometric and meristic differences between S. melanotheron and C. guineensis. Together, C. guineensis might then be present in other isolated areas of the Atlantic Sahara (Fig. 5). Further sampling is needed to describe the distribution range of this species, which would have important implications both in terms of conservation and evolutionary biology. There is large polychromatism within S. melanotheron sampled in PNBA, which can either present a silvery colour pattern or a totally black one, whereas the original species description only mentioned some black spots in adults (Teugels & Thys van den Aude- naerde, 2003). Polychromatism may have a different origin in these cichlids, such as sexual dichromism or social communication (Maan & Sefc, 2013). In S. melanotheron, colour pattern variations are probably related to sexual maturity as black spots are often larger and darker in mature males and absent in juvenile ones (Teugels & Thys van den Audenaerde, 2003).

COPTODON GUINEENSIS: A SPECIES COMPLEX The systematics of Cichlidae has a long history, and many successive revisions have been performed, but recent molecular phylogenies call for a new one (Nagl et al., 2001; Klett & Meyer, 2002; Schwarzer et al., 2009; Dunz & Schliewen, 2013). Among the most important results of these molecular phylogenetic reconstructions is the demonstration that Tilapia sensu lato is not a monophyletic group, but consists of several genera included in 10 lineages (Dunz & Schliewen, 2013). At the species rank, that study provided limited insights into diversity. While Dunz & Schliewen (2013) mentioned the presence in the Coptodonini tribe of 31 species distributed into three clades, they only briefly mentioned the presence of cryptic species of C. zillii, C. guineensis or Coptodon louka (Thys van den Audenaerde 1969), by naming some taxa as C. sp. aff. zillii, C. sp. aff. guineensis or C. sp. aff. louka. In this study, all phylogenetic reconstructions with all molecular markers (nuclear or mitochondrial) reveal C. guineensis to be paraphyletic and to consist of three to four evolutionary lineages embedded in two clades consisting of all species of Coptodon, with the exception of C. tholloni and Coptodon congica (Poll & Thys van den Audenaerde 1960). Consequently, C. guineensis is a species complex and, considering the distribution range of these lineages [Fig. 5(a)], these putative species are parapatric. Thus, the lineage representing the valid C. guineensis may correspond to the Cg-I lineage (Fig. 4) as its distribution ranges includes the type locality of C. guineensis, i.e. the Ghanaian Ashantee area (Bleeker in Günther, 1862). Coptodon guineensis is a euryhaline species with a clear affinity for marine or brackish water, as demonstrated by the origin of the current samples, which were collected in the Aby Lagoon in Côte d’Ivoire and Weija Lake in Ghana, which have salinities close to marine (Asante et al.,

2008). Nevertheless, it is phylogenetically extremely close to the parapatric freshwater species, C. dageti and C. discolor (Teugels & Thys van den Audenaerde, 2003). This phylogeographic pattern suggests a recent origin of C. dageti and C. discolor, which would have diverged from the ancestral C. guineensis when entering freshwater environments. Other lineages of C. guineensis highlighted in the phylogenetic trees correspond to undescribed species or species that were invalidated after successive revisions. In Sene- gal, two species were described (Tilapia affinis Duméril 1861 and Tilapia polycentra Duméril 1861) that were later synonymized with C. guineensis (Eschmeyer, 2015). To determine which species name should be resurrected, a more complete redescription of each type species has to be performed, which is beyond the scope of this study. Among all lineages of C. guineensis highlighted in this study, the Mauritanian/Senegalese one (Cg-II in Fig. 4) is the closest to C. guineensis sensu stricto as it shares both the same mitochondrial clade [CII in Fig. 3(b)] and ecological niche (coastal environments). This species, however, has close phylogenetic relationships with freshwater species such as C. camerunensis and C. sp. aff. louka ‘Samou’, justifying its taxonomic status as a valid species. The two other lineages from Nigeria and Gabon, identified morphologically as C. guineensis, share the same clade with C. rendalli and C. zillii [CI within Fig. 3, see also Fig. 4(a)] and are not closely related to C. guineensis. Coptodon rendalli and C. zillii have the widest distributions among all species of Coptodon; whereas C. zillii is known from Morocco (Qninba & Mataame, 2009) to the Democratic Republic of Congo and the Middle East (Teugels & Thys van den Audenaerde, 2003). Coptodon rendalli is not naturally distributed in West Africa although it is very common in aquaculture and widely distributed due to anthropogenic activities (Lazard & Rognon, 1997). The CI clade also includes species with restricted distribution ranges including species belonging to Cameroonian species flocks. With the exception of these latter species, distribution ranges of C. zillii and C. rendalli appear to be allopatric [Fig. 5(b), (c)], suggesting that vicariant events relating to landscape evolution of African rivers certainly played an important role in the divergence and evolution of Coptodon. In conclusion, this study sheds new lights on C. guineensis, which occurs along the West African shoreline as a complex of species, each with a narrow distribution range. More phylogenetic investigations are needed to describe the species diversity inside the C. guineensis complex, and determine the evolutionary history underlying the diversity of Coptodon. If ecological speciation occurred in Coptodon, as suggested by phylogenetic investigations of Cameroonian crater lake cichlids, vicariance is also an important evolutionary force that needs to be evaluated by further studies.

The authors are grateful to M. M. Dia for his help and support during the sampling, U. K. Schliewen for some samples used in this study and F. Leprieur for advice and help with statistical analysis. Data used in this work were (partially) produced with the molecular genetics analysis facilities of the Centre Méditerrannéen de l’Environnement et de la Biodiversité. We are grateful to F. Cerqueira for her technical support in the laboratory. This study was funded by Institut de Recherche pour le Développement (IRD) and Gesellschaft für Internationale Zusammenarbeit (GTZ). J.C.B. is supported by Fundação para a Ciência e Tecnologia, FCT (IF/00459/2013). Fieldwork was partially supported by FCT (PTDC/BIA-BEC/099934/2008) through the EU programme COMPETE. The authors are indebted to D. McKenzie and P.Chakrabarty for editing of the manuscript.

Supporting Information Supporting Information may be found in the online version of this paper: Table SI. Cichlid samples used in COI and CR phylogenetic analyses. Sample code, species, location, collector name and GenBank accession number are noted

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