Genetic characterization of the Aphyosemion calliurum species group and description of a new species from this assemblage: A. campomaanense (Cyprinodontiformes: Aplocheiloidei: Nothobranchiidae) from Southern Cameroon Author Agnèse, Jean-François Author Brummett, Randall Author Caminade, Pierre text Zootaxa 2009 2045 43 59 journal article 10.5281/zenodo.186435 73a334ca-48c1-4ce9-8fcc-59ce4a8bc213 1175-5326 186435 Aphyosemion campomaanense new species ( Figs. 4–10 ; Table 2 ) Holotype . MRAC A7-30-P-1, male, 36 mm SL ( Fig. 4 ); Cameroon : Campo-Ma’an National Park, small stream under forest cover; 2.33735° N , 10.20435° E ; J.-F. Agnèse, R. Brummett & P. Caminade, 21 January 2006 . Site ABC 06/86. Paratypes . MRAC A7-30-P-2-21, 9 males , 26–40 mm SL and 11 females 33–47 mm SL ( Fig. 5 represents one of the females); same data as holotype . MRAC A7-30-P-22-24, 2 males 27-38 mm SL and one female 35 mm SL; Campo-Ma’an National Park, small stream under forest cover; 2.33372° N , 10.20322° E ; J.-F. Agnèse, R. Brummett & P. Caminade, 20 January 2006 . Site ABC 06/84. MRAC A7-30-P-25-26, 2 males 31–34 mm SL; Campo-Ma’an National Park, small stream under forest cover; 2.34210° N , 10.16753° E ; J.-F. Agnèse, R. Brummett & P. Caminade, 20 January 2006 . Site ABC 06/83. MNHN (2007- 1533-1534 ) one male and one female, 41–43 mm SL; Campo-Ma’an National Park, small stream under forest cover; 2.34210° N , 10.16753° E ; J.-F. Agnèse, R. Brummett & P. Caminade, 20 January 2006 . Sampling reference ABC 06/83. MNHN (2007-1535) one male, 31 mm SL; Campo-Ma’an National Park, small stream under forest cover; 2.35508° N , 10. 15188° E; J.-F. Agnèse, R. Brummett & P. Caminade, 21 January 2006 . Site ABC 06/85. MNHN (2007- 1536-1541 ) three males 30–39 mm SL and three females, 30–55 mm SL; Campo- Ma’an National Park, small stream under forest cover; 2.34790° N , 10.16948° E ; J.-F. Agnèse, R. Brummett & E. Kornobis, 24 January 2007 . Site ABK 07/179. MNHN (2007- 1542-1545 ) three males 29–39 mm SL and one female, 29 mm SL; Campo-Ma’an National Park, small stream under forest cover; 2.33270° N , 10.19335° E ; J.-F. Agnèse, R. Brummett & E. Kornobis, 24 January 2007 . Site ABK 07/180. FIGURE 4. Holotype of Aphyosemion campomaanense , MRAC A7-30-P-1, adult male (36 mm SL) from site 21 (Fig. 1) in the Campo-Ma’an reserve (ABC 06/86, 2.33735° N, 10.20435° E). FIGURE 5. Paratype of Aphyosemion campomaanense , MRAC 07-30-P-2-21, adult female (34 mm SL) from type locality (site 21, Fig. 1). Diagnosis. Aphyosemion campomaanense is distinguished from congeners by a combination of coloration characteristics. The new species is distinguished from A. ahli ( Fig. 11 ) by a much more pronounced and darker blue background color than any A. ahli population (Fig. 64 & 65, Pl. 50) in Amiet (1987) . The background color is here defined as the color that covers the majority of the body. In addition, A. ahli has a caudal fin with symmetric coloration while in A. campomaanense the caudal fin coloration is asymmetrical with a yellow lower margin and a white upper margin (vs. red and yellow margins in A. ahli ). Although such asymmetric caudal fin coloration is occasionally observed in A. ahli (the upper margin sometimes being reduced to only the red part), the red and yellow lower margin is always present. In A. campomaanense , this margin is only yellow. Finally, the pectoral fins in A. campomaanense are deep orange vs. pale yellow in A. ahli . Aphyosemion campomaanense is distinguished from A. lividum , Fig. 12 , by striped dorsal and anal fins vs. unstriped fins; and red dots aligned like vertical stripes posteriorly versus no red punctuation posteriorly. FIGURE 6. Aphyosemion campomaanense , adult male from ABC 06/83 (site 20, Fig. 1). This estimated 14 month old male never had any yellow margin on the anal fin, nor did any other male sampled from of this population. Specimen not preserved (approximately 40 mm SL). Photo by J.-F. Agnèse. FIGURE 7 . Aphyosemion campomaanense , adult male from ABK 07/181 (site 20, Fig. 1). This male (estimated 10 months old) has an incomplete anal fin margin. Specimen not preserved (approximately 40 mm SL). Photo by P. Delaroche The new species is distinguished from A. edeanum by an obvious blue vs. reddish background color; asymmetric caudal fin coloration with a yellow lower margin and a white upper margin vs. red and yellow margins (as in A. ahli ); and deep orange pectoral fins vs. transparent or pale blue. Aphyosemion campomaanense can be distinguished from A. heinemanni by a more pronounced blue background color; asymmetric caudal-fin coloration with a yellow lower margin and a white upper margin vs. a semicircular red sub-margin on a rounded caudal fin; and the presence of red dots on the anal fin vs. no dots. Aphyosemion campomaanense can be distinguished from A. pascheni pascheni and A. pascheni festivum by the presence of vertically aligned red dots in the posterior vs. scattered weak red spotting; red dots on the anal fin vs. no dots; red stripes on the dorsal fin versus no stripes; yellow lower margins vs. white on the anal and caudal fins; and red vertical stripes on the caudal fin vs. a series of red dots ( A. pascheni pascheni ) or straight lines or series of dots along rays ( A. pascheni festivum ). FIGURE 8 . Aphyosemion campomaanense , adult male from ABK 07/181 (site 20, Fig.1, identical to ABC 06/84). This male was captured in January 2007 when estimated to be two month old. Left and right profiles were photographed in March (estimated 4 months old, approximately 30 mm SL), May (estimated 6 months old) and September 2007 (estimated 10 months old, approximately 40 mm SL). The yellow margin of the anal fin has quite completely disappeared after 10 months. The caudal pattern also changed during this period in terms of number and shape of the vertical bars. One can also observe the asymmetrical pattern of the right and left sides (especially the pattern of bars on the posterior). Specimen not preserved. Photos by A. Persson. Description . See Figs. 4–10 for general appearance of A. campomaanense , Table 2 for morphometric data of holotype and paratypes . Body and fin morphology. There is little sexual dimorphism in body form and allometry in body growth (visually checked on the data matrix) in A. campomaanense . The dorsal-fin deviation to anal fin (D/A) is the same in male and female: towards the rear, closer to the end of the anal (usually D/A= 6–7). Dorsal and anal fin shapes in the male and female differ only by the male fins being wide and triangular while the female fins are rounded. In contrast, caudal fin sexual dimorphism is strong with a lyre shape with extended filaments in the male and a round shape in the female. Meristic characters. Measurement data are summarized in Table 2 , (D=10.73, A=14, D/A=6.84). These are not diagnostic and are within the average values reported for other species of the A. calliurum species ( Huber, 2000 ). FIGURE 9 . Aphyosemion campomaanense , adult female from ABK 07/181 (site 20, fig. 1, identical to ABC 06/84). Specimen not preserved (approximately 30 mm SL). Photo by A. Persson. FIGURE 10 . Aphyosemion campomaanense , adult male from ABK 07/181 (site 20, fig. 1). This male (estimated 12 months old, approximately 45 mm SL) still has a large yellow margin to the anal fin. Specimen not preserved. Photo by J.-F. Agnèse. FIGURE 11 . Aphyosemion ahli , adult male from ABC 06/79 (site 11, Fig. 1). Specimen not preserved (approximately 35 mm SL). Photo by J.-F. Agnèse. FIGURE 12 . Aphyosemion lividum , adult male from site 6 (ABC 06/94). Specimen not preserved (approximately 30 mm SL). Photo by J.-F. Agnèse. Coloration of live males. The dorsal surface is light brown. The head, laterally pigmented with light blue has three oblique red bars on the operculum, a sub-ocular red line and anterior and posterior infrabuccal red bands. The flanks vary from light blue to deep blue with a metallic sheen. Two or three horizontal lines of discontinuous red dots extend along the forequarter of the flanks. Towards the posterior, red dots merge progressively to create large irregular vertical bars. The dorsal fin is blue with dense red stripes forming oblique lines transverse with respect to the rays. The pectoral fins are orange. The anal fin is light blue-green with red stripes forming oblique lines transverse with respect to the rays. On the anal fins of young males are three superimposed longitudinal marginal bands of red, white and yellow. The external yellow band is not always present; this point will be detailed later in the text under “distribution and variability”. The caudal fin coloration is asymmetric. Background coloration is light blue with broad vertical red bands, which exhibit high individual variation in terms of number and shape (interrupted or not). The upper margin is white with one to three rays often extending to form a long extension. The lower margin is yellow with a sword-like extension. The fin coloration pattern on the pelvic fins is identical to that of the anal. Coloration of live females. The females have a grayish yellow body on which two horizontal bands of red dots are present on the anterior third of the body and narrow brown vertical stripes on the posterior twothirds. Two or three oblique red bands are present on the operculum. All fins except pectorals have pale yellow glints. Red-brown inter-radial dots are densely and regularly spread throughout the dorsal and the caudal fins. On the caudal fin, these dots are distributed in concentric arcs. The anal fin shows traces of interradial stripes. The pelvic fins are edged with light blue glints. Coloration of ethanol preserved males and females. Body light grey in the posterior part and around the belly to dark grey in the dorsal area. All red spots of live specimens remain. The yellow color in the male caudal-fin turns to white. The pectoral fins are transparent in both sexes. Karyotype. Two specimens were karyotyped, one male (population ABC 06/83, site 18, Fig. 1 & Table 1 ) and one female (population ABC 06/84, site 20, Fig. 1 & Table 1 ). More than 20 metaphases per specimen were analyzed to determine the diploid number and 15 complete karyotypes were made to classify the chromosomes according to their morphology. No differences were found between the male and female karyotypes (i.e., morphologically differentiated sex chromosomes are absent). A. campomaanense was characterized by a diploid number of 44 chromosomes and a fundamental number (of arms) of 58. Chromosomes were classified according to their morphology ( Levan 1964 ): 15 pairs of acrocentric chromosomes almost equivalent in size, 6 pairs of subtelocentrics decreasing in size, and one pair of short heteromorphic chromosomes, (one submetacentric and one metacentric; Fig. 13 ). Further analysis is required to determine if the observed size polymorphism is due to different amounts of heterochromatin as commonly observed in fish chromosomal studies ( Völker et al ., 2007 ). Such a chromosomal formula, with 44 chromosomes, has until now never been observed in any of the A. calliurum species group, with A. edeanum ( A. ahli from North of the Nyong River in Scheel, 1990 ) previously identified as the species with the highest number of chromosomes, 2n=40 and NF=58, Scheel (1990) . FIGURE 13. Metaphase (A) and karyotype (B) of a female Aphyosemion campomaanense from ABK 07/181 (site 20, Fig. 1) (2n=44, NF=58) showing 1 pair of metacentric (with different amounts of heterochromatin), 6 pairs of subtelocentric and 15 pairs of acrocentric chromosomes. Phylogenetic relationships. The 29 sequences obtained (GenBank references EU272795 to EU272816 and EU885232 to EU885237 ) permitted the construction of a phylogenetic tree ( Fig. 3 ) representing the relationships between A. campomaanense and its closest congeners. Specimens of A. campomaanense coming from six different locations all grouped together in a highly supported clade. This assemblage was clearly differentiated from all the other species and particularly from A. ahli and A. lividum . Taking into account that all A. ahli haplotypes (largely representative of the overall variability of the species throughout its range) are grouped together and that A. edeanum occupies an intermediate position between A. ahli and A. campomaanenese , the new species cannot be part of the A. ahli gene pool. It is also apparent from the tree that A. campomaanense is genetically distinct from A. lividum . These observations indicate that A. campomaanense is a distinct new species. Distribution and vaiability. The complete distribution of A. campomaanense is not well known because a large zone north of the Campo-Ma’an National Park still has not been sampled, mainly because no road or even track exists. A. campomaanense has been found in 7 locations, 5 of them inside the Campo-Ma’an National Park where it occupies the western part up to point ABC O 6/86 ( 2.33735° N , 10.20435° E ). To the east and north, the species is replaced by A. cameronense (Boulenger, 1903) . The species is also present in the vicinity of Nkoelon (ABC 05/55, 2.37042° N , 9.95850° E ), a few kilometers west of the park and also a few kilometers northward (ABK 07/183, 2.43089° N , 10.11654° E ) outside of the park. Up to now, this species has not been collected south of the Ntem River in Equatorial Guinea , but its presence there cannot be ruled out. Unlike A. ahli ( Fig. 11 ) or A. edeanum , which show significant inter-population morphological variability ( Amiet, 1987 ), A. campomaanense populations vary only in regard to some minor differences in anal and caudal fin coloration. For example, males from the population ABC 06/83 (site 18, Fig. 1 ) exhibit no yellow band in the anal fin ( Fig. 6 ). In some male specimens of population ABK 07/181 (site 20, Fig. 1 ) the yellow band can disappear partially ( Fig. 7 ) or totally ( Fig. 8 ) with age (between the ages of six months and one year). Males of other populations keep their yellow anal fin band throughout their life ( Fig. 10 ). Among individuals within a population, caudal fin coloration varies in the number (usually 3– 5) and shape (interrupted or not) of the large red vertical bands, which can be used as fingerprints to identify individual males. These bands also vary with age ( Fig. 7 ). Ecology. Like most of the A. calliurum species group, A. campomaanense is usually found in small streams and shallow pools in the rainforest. They usually stay close to the banks, avoiding the deepest and fastest water. Among killifish, A. campomaanense is sympatric with Aphyosemion (Chromaphyosemion) lugens Amiet, 1991 , Epiplatys infrafasciatus (Günther, 1866) and Procatopus nototaenia Boulenger, 1904 . Etymology. From Campo-Ma’an, in reference to its origin in the Campo-Ma’an National Park, Southwestern Cameroon .