Aphyosemion (Chromaphyosemion) pamaense, Agnese, Jean-Francois, Legros, Olivier, Cazaux, Benoite & Estivals, Guillain, 2013

Agnese, Jean-Francois, Legros, Olivier, Cazaux, Benoite & Estivals, Guillain, 2013, Aphyosemion pamaense, a new killifish species (Cyprinodontiformes: Nothobranchiidae) from Cameroon, Zootaxa 3670 (4), pp. 516-530 : 521-528

Aphyosemion (Chromaphyosemion) pamaense , sp. nov.

Figs. 2–5 , Table 2

Holotype. MRAC B2-08-P-1. Adult male, 29.7 mm SL (38.4 mm T.L.), Cameroon, 1.6 km east of Pama village in the direction of Béla. Small brook forming the upper part of the river Pama, Nyong basin, 03° 16.420 N, 10° 05.235 E, 24 Feb. 2010, Field number code ADK-10-323, collectors Jean-François Agnèse, Cyrile Dening, Grégoire Kayoum.

Paratypes. MRAC B2-08-P-2. Adult female, 27.9 mm SL, collected with the holotype. MRAC B2-08-P-3-20. One female and 17 males, 20.6 –28.4 mm SL.

Non types: MRAC B2-08-P-21-27. Seven juveniles, collected with the holotype.

Diagnosis. Aphyosemion pamaense belongs to the subgenus Chromaphyosemion Radda, 1971 , as it is characterized by the presence of two dark lateral bands in both sexes, these bands may disappear in males depending on the mood of the fish. The caudal fin is shaped with pronounced dorsal and ventral streamers in males. Streamers are also present in the dorsal and anal fins. The species is also characterized by 9–14 dorsal rays, and 11– 16 anal rays.

The new species ( Figs. 2–5 ) differs from all other Chromaphyosemion species by the following combination of characters observed in males: orange background of the unpaired fins, anal fin without any spots, orange throat, purple to blue-grey flanks.

The new species differs from A. splendopleure and from A. volcanum by the head and front of the body that are orange versus yellow; blue-grey to violet flanks with few red dots on scales versus blue to pinkish flanks with five or six lines composed of red dots from the post opercular zone to the caudal peduncle; unpaired fins totally orange versus yellow-green with sometimes an orange zone in the anal and caudal fins (Male in Fig. 2 is young and its unpaired fins are not yet fully orange).

In A. alpha Huber, 1998 , the dorsal fin is orange and the anal one blueish, in A. malumbresi , dorsal is orange and anal whitish versus anal and dorsal orange in A. pamaense .

A. pamaense is distinguished from A. koungueense by yellow-green unpaired fins, lateral bands that are often visible and sometimes very dark, pinkish-beige flanks in the later versus orange fins and grey and barely present lateral bands and blue-grey in the former.

In A. bitaeniatum , a rather variable species, the fins are orange but the anal has a bluish background with a more or less extended orange area versus totally orange for the new species. The upper and lower lobes of the caudal fin are orange; including the acumens versus a small portion of the lower lobe is orange with yellow streamers. Sides of A. bitaeniatum are cool blue versus grey to violet in A. pamaense .

A. pamaense is distinguished from A. omega by the orange coloration of the throat, periopercular region and edge of the gills versus grey to blackish; a lesser importance of the orange colour in the caudal fin versus an orange zone developed in the upper and lower lobes; translucent pectoral fins vs. orange fins; blue grey to violet flanks versus grey to orange flanks; an orange head versus a pearly white to yellowish one; yellow streamers versus orange.

The anal fin of A. pamaense is totally orange without spots versus spotted or stained in the following species of Chromaphyosemion : A. bivittatum has a spotted anal fin with a blue background and sometimes an orange area, blue grey flanks with an orange throat, with many brown dots in the dorsal area; A. poliaki has a spotted anal fin with blue grey background, brown sides with a dorsal area showing 3 or 4 rows of golden scales; A. riggenbachi has a stained anal with a blue yellow background, blue grey flanks with red dots; A. loennbergii has a stained anal with a blue background and an orange area, sometimes the anal fin is entirely orange, blue violet or orange flanks with rows of red dots; A. punctulatum has a highly spotted anal fin with a blue green background, the dorsal area is brownish, flanks are pearl white; A. lugens and A. melinoeides have a turquoise or gold stained anal fin, flanks are pale green with many dark dots in the dorsal area, the throat is orange for A. melinoeides ; A. kouamense has a spotted yellow green anal fin, an orange throat, blue grey or green grey flanks; A. melanogaster has a yellow green anal fin with few spots, the dorsal area is brownish and the flanks are beige; A. malumbresi has a whitish anal fin with spots, bluish sides, a dorsal area brown with rows of orange scales; A. ecucuense has a spotted anal fin with an orange background, sometimes blue green with an orange area, pale grey to beige sides; A. erythron has a spotted orange anal fin, sides are pale grey to beige, all scales on the sides have red dots

Description. See Figs. 2–5 for general appearance of A. pamaense , Table 2 for morphometric data of holotype and paratypes.

Coloration of live males. The anterior part of the body, from the lower jaw to the pelvic fins, is orange ( Fig. 2 ). This colouration tapers at the beginning of the anal fin. The lower lip is bluish, the area below the eye is purplish blue with a thin red horizontal line. The opercular spot is barely visible while there are three oblique and sinuous red lines just behind the eye. Sides are blue-grey, blue or purple depending on the mood or light intensity. There are two rows of paradorsal red scales from the operculum to the caudal peduncle on a yellow bright orange background. Two other less visible red lines are on the flanks, from the operculum to the mid body. The ventral region close to the caudal peduncle is underlined with 12 or 13 red dots. The dorsal fin has an orange centre on a greenish background. There are rows of carmine red spots between the rays and streaks close to the edges, especially at the apex of the posterior side are visible. Anal fin is intense orange without any spots with a blue marginal band and a red submarginal band topped with a very fine yellow green band. The background of the tail is yellow-green, distally blue. Upper and lower lobes are orange in the adult specimen. Very long red small streaks are present starting from the caudal fin base to the edges. Four to five red spots are barely visible at the base of the peduncle. A submarginal red band and a blue marginal band are present on the lower margin. Acumens at the apex of the unpaired fins are orange yellow. Pectoral fins are translucent with blue white reflections on the edges. The colour pattern of pelvic fins is identical to that of anal fins an orange background with a red submarginal band and a blue marginal band. The two dark lateral stripes are rarely seen in males of this species.

Coloration of live females. The body is brownish with five or six rows of red dots from post-opercular area to the caudal peduncle ( Fig. 3 ). The ventral region is orange-pink with blue-grey highlights on the sides. Three red and oblique sinuous lines in the opercular area are visible. The lower jaw is blue grey. There is a red line below the eye. Dorsal fin is yellow green with lines of red dots between the rays. Anal fin is bluish with yellow-green reflections in the anterior part, three to four red dots hardly visible. Caudal fin is greyish-blue with six or seven small red streaks on the median part of the fin from the caudal fin base to the edges. The dorsal part has six rows of red spots between the rays. The ventral part, is yellowish and without any spots. Pectorals are translucent, with blue white reflections on the edges.

Coloration of ethanol preserved males and females. Males dorsal part of the body is grey from the head to the caudal peduncle ( Fig. 4 ). The ventral part is yellowish beige with more yellow on the belly. Two horizontal lines of red spots run from the opercular region to the caudal peduncle. A third line is visible but incomplete below. Behind the eye, three oblique red sinuous lines are present. The anal fin is orange without spots. The caudal fin is blue-grey with seven long red narrow streaks in the central part. There are few small red streaks in the upper lobe, which has a slightly orange background. The lower lobe is also slightly orange. The dorsal fin is orange with black dots. The pectoral fins are greyish and the pelvic fins orange. Streamers of unpaired fins are translucent. On paratypes, two grey horizontal lateral lines are visible, but not in the holotype.

Females body is greyish with beige ( Fig. 5 ). Five more or less complete lines of red dots run from the eye to the caudal peduncle. Two sinuous red lines are present behind and below eye. The anal fin is whitish without spots but darker at the base. The pigmentation pattern of the caudal fin consists of six dark red flames with a few spots between the rays. The upper and the lower parts are punctuated with dark dots. The dorsal fin has brown spots between the rays. The pectoral and pelvic fins are whitish. Two grey horizontal lateral bands are present in the two paratypes examined.

Distribution and habitat. Aphyosemion pamaense is only known from the type locality in the surroundings of Pama, from the head of the Pama River, which belongs to the Nyong basin. It is a small stream in the forest, about 3 m wide and 0.4 m deep with a muddy or sandy bottom (Figs. 6 & 7). At the end of the dry season (generally in February) the stream stops flowing and the water turns green due to algae growth. The other Cyprinodontiforms species that have been found in syntopy with A. pamaense are Epiplatys infrafasciattus (Günther, 1866) , Aphyosemion edeanum Amiet, 1987 , and Procatopus similis Ahl, 1927 . All these species have a larger distribution.

Genetic characterisation. A 1028 base-pair alignment for cytochrome b region from the mitochondrial genome was obtained after trimming the ends of each sequence. A total of 68 specimens were sequenced. One specimen per out-group species was sequenced while for Chromaphyosemion species, two specimens per populations were sequenced. These two specimens usually have identical haplotypes except for population 3 were two different haplotypes (differing by one mutation) were found. In specimens from populations 13 and 15 we found the same haplotype. A total of 426 variable sites were identified, from which 367 were parsimony informative (i.e. shared by at least two different sequences).

FIGURE 6. The type locality of Aphyosemion pamaense is a small stream in the forest, about 3 m wide and 0.4 m deep with a muddy or sandy bottom. Photo taken the 22nd of January 2007.

FIGURE 7. The type locality at the end of the dry season (generally in February) when the stream stops flowing and the water turns green due to algae growth. Photo taken the 24th of February 2010.

Using the Bayesian Information Criterion (BIC) in MEGA ( Tamura et al., 2011), the optimal model of sequence evolution was TN93+G. This model ( Tamura and Nei, 1993) accounts for the difference between transitions and transversions and differentiates the two kinds of transitions. The G parameter indicates that nonuniformity of evolutionary rates among sites is modelled by using a discrete Gamma distribution. This model has been used for the subsequent analysis. The three different methods used, Maximum Likelihood, Distance and Minimum Evolution, gave congruent results, summarised in Figure 8 . The different trees were rooted using Aphyosemion franzwerneri , A. ahli , A. exiguu m, A. elberti , A. amoenum , and A. cameronense .

All Chromaphyosemion species appeared to be monophyletic as previously observed ( Collier, 2006; Agnèse et al. 2006). Phylogenetic relationships within Chromaphyosemion were also congruent with preceding studies ( Collier, 2006; Agnèse et al. 2006). Aphyosemion alpha occupied the more basal position within Chromaphyosemion followed by A. lugens and A. melinoeides grouped together. All the other Chromaphyosemion species were clustered in a highly supported group with A. bivittatum and A. bitaeniatum occupying a basal position. In this group, A. kouamense , A. ecucuense , A. erythron , A. malumbresi and A. melanogaster were clustered together. This cluster was the sister group of all the other Chromaphyosemion from Cameroon in which A. pamaense took place. A. pamaense was the sister taxon of a group of four species: A. punctulatum , A. kougueense , A. omega and A. splendopleure .

Karyotypes. Six specimens were karyotyped, three males and three females. For each individual, 15 metaphases were observed to determine the karyotype. This species is characterized by a diploid number of 2n=35 for males ( Fig. 9 ) and 2n=36 for females ( Fig. 10 ). Sex chromosomes were morphologically differentiated. Males showed two pairs of large metacentric chromosomes, three small metacentric chromosomes, two submetacentric (one large and one small chromosome) and 13 pairs of acrocentric chromosomes ( Fig. 9 ). Females showed two eª

eb A. splendopleure 97/9e/98 4

ƽ A. omega 99/99/99 6

90/88/88 17

8

84/89/ A. koungueense 7

60/76/61 10

20 A. punctulatum

99/99/99 16 A. pamaense sp. nv.

1 A. volcanum A. poliaki 98/98/99

18

100/98/99 19

9

58/59/49 12 A. loennbergii 11

1e

14

1ƽ

A. riggenbachi

A. kouamense 99/99/97 21 A. melanogaster A. erythron A. ecucuense 999//9999//99999 A. malumbresi 99/99/99

A. bivittatum

86/91/74 A. bitaeniatum

100/99/100 A. lugens

A. melinoeides

A. alpha

A. franzwerneri

A. ahli

100/100/100 A. exiguum

A. elberti 81/84/82 100/100/100 A. amoenum

A. cameronense

0.05

pairs of large metacentric chromosomes, two pairs of small metacentric chromosomes, two submetacentric chromosomes and 13 pairs of acrocentric chromosomes ( Fig. 10 ). The difference between sexes could result from the fusion of one small metacentric and one submetacentric chromosome to form the large submetacentric chromosome in the male.

Ethymology. This species is named after the river and the nearby village Pama, the collection locality of the types.

Figures

FIGURE 2. Wild male of Aphyosemion pamaense. Specimen from the same locality as the holotype, not preserved. Photograph by R. Pohlmann.

FIGURE 3. Wild female of Aphyosemion pamaense. Specimen from the same locality as the holotype, not preserved. Photograph by R. Pohlmann.

FIGURE 4. Aphyosemion pamaense, holotype, preserved in ethanol.

FIGURE 5. Aphyosemion pamaense, MRAC B 2 - 08 - P- 2, female paratype, preserved in ethanol.

FIGURE 8. Consensus tree based on Distance method (Kimura 2 and Neighbor Joining methods), Maximum Likelihood, Minimum Evolution. Numbers above the branches are percentages of bootstrap values based on 2,000 replicates for each method. Numbers correspond to sample location (Figure 1 and Table 1). When more than one sequence were found from the same location, they have been noted a, b. Scale refers to Kimura 2 distance.

FIGURE 9. Karyotype of a male Aphyosemion pamaense. (2 n = 35, NF = 44). Chromosomes were stained with Giemsa. It consists of two pairs of large metacentric chromosomes, 3 small metacentric chromosomes, two submetacentric (one large and one small chromosome) and 13 pairs of acrocentric chromosomes. Scale bar = 10 µm.

FIGURE 10. Karyotype of female Aphyosemion pamaense (2 n = 36, NF = 46). Chromosomes were stained with Giemsa. It consists of two pairs of large metacentric chromosomes, two pairs of small metacentric chromosomes, two submetacentric chromosomes and 13 pairs of acrocentric chromosomes. Scale bar = 10 µm.

Tables

TABLE 2. Meristic and morphometric data of A. pamaense. N, number of specimens studied; HT Holotype values; Min, minimum observed value; Max, maximum observed value; Mean, mean of observed value; SD, standard error; D, Dorsal-fin rays; A, Anal-fin rays; D / A = displacement of the dorsal in relation to the anal fin expressed as the number of anal fin rays; LL, lateral line scale rows; pDor, predorsal scales; TRAV, transversal scales; S. L. = standard length in mm; all other mesurements in percentage from Standard Length. T. L. = total length; P. D. = predorsal distance; P. A. = preanal distance; P. V. preventral distance; Ht, height at the anal; dcp, caudal peduncle height; Hd, head length; I. O., interorbital space; Eye, eye diameter.

Character	N	HT	Min	Max	Mean	SD
D	20	12	10	13	11.40	0.82
A	20	13	12	15	13.20	0.77
D/A	20	0	0	3	0.65	0.88
L.L.	20	27+2	25 (+1)	28 (+3)	25.95 (+2)	0.89 (+0.73)
pDor	20	13	13	14	13.35	0.49
TRAV	20	7	7	10	8.00	0.86
S.L.	20	29.7	29.7	20.6	25.06	2.67
T.L. (%)	20	130	132	119	127.37	4.27
P.D. (%)	20	62	77	57	64.22	4.62
P.A. (%)	20	62	66	53	61.0 2	3.12
P.V.(%)	20	46	51	42	46.47	3.0 1
Ht (%)	20	22	22	17	19.35	1.55
dcp (%)	20	12	15	10	11.73	0.99
Hd (%)	20	28	33	26	29.88	2.0 3
I.O. (%)	20	14	15	12	13.12	0.89
Eye (%)	20	9	10	7	8.45	1.08