Petrocephalus boboto and Petrocephalus arnegardi, two new species of African electric fish (Osteoglossomorpha, Mormyridae) from the Congo River basin

Abstract A specimen of the African weakly electric fish genus Petrocephalus (Osteoglossomorpha, Mormyridae) collected in the Congo River at Yangambi, Orientale Province, Democratic Republic of Congo, is described as a new species. Petrocephalus boboto sp. n. can be distinguished from other Central African species of Petrocephalus by a combination of the following characteristics: three distinct black spots on the body, one at the origin of the pectoral fin, one at the origin of the caudal fin and one below the anterior base of the dorsal fin; Nakenrosette and Khelrosette electroreceptor clusters distinct on head but Augenrosette cluster reduced in size; 23 branched dorsal rays, 34 branched anal rays, and electric organ discharge waveform triphasic. Petrocephalus boboto sp. n. most closely resembles the holotype of Petrocephalus binotatus but is easily distinguished from it by its smaller mouth. A comparative molecular analysis including 21 other Petrocephalus species shows Petrocephalus boboto sp. n. to be genetically distinctive and to represent a deep lineage in the genus. Two specimens of Petrocephalus collected at Yangambi are morphologically similar and genetically closely related to specimens previously assigned to Petrocephalus binotatus, collected in the northwestern Congo River basin within Odzala-Kokua National Park, Republic of the Congo. This prompts us to formally describe a new species from these collections, Petrocephalus arnegardi sp. n., that, although similar to the holotype of Petrocephalus binotatus, can be distinguished from it by its smaller mouth and shorter interorbital width.


Introduction
The monophyletic African weakly electric fishes superfamily Mormyroidea (Teleostei, Osteoglossomorpha) contains two families, the Gymnarchidae and Mormyridae, and 212 species (Eschmeyer and Fong 2014). All mormyroids generate and sense weak electric discharges for the purpose of intraspecific electrocommunication and spatial electrolocation using complex electrogenic and electroreceptive organ systems (Moller 1995, Turner et al. 1999, Bullock et al. 2005. While mormyroids are immediately identifiable by their distinctive appearance, considerable morphological, electrophysiological and behavioral diversity has evolved within the group (Arnegard et al. 2010b, Rabosky et al. 2013). These differences lead to the recognition of three main lineages: the monotypic Gymnarchidae and the two reciprocally monophyletic mormyrid subfamilies Mormyrinae and Petrocephalinae with 168 and 43 valid species, respectively (Taverne 1972, Sullivan et al. 2000, Eschmeyer and Fong 2014.
Recent comparative studies have revealed significant electrophysiological differences between Mormyrinae and Petrocephalinae (Lavoué et al. 2008. In particular,  suggested that differences among lineages in central and peripheral electrosensory anatomies are correlated with higher electric organ discharge (EOD) waveform diversity and greater species richness in Mormyrinae relative to the Petrocephalinae. While Mormyrinae are clearly more speciose than Petrocephalinae, true species diversity is clearly underestimated in both groups. Despite often subtle morphological differentiation among species of Petrocephalus, the sole petrocephalin genus, the pace of new species discovery and description has been rapid in recent years, with about one third of Petrocephalus diversity having been described in the 21th century (Lavoué et al. 2004, 2010, Lavoué 2011, 2012, Kramer et al. 2012. Petrocephalus currently includes 43 valid species (Eschmeyer and Fong 2014), not including Petrocephalus balteatus (for justification, see Daget 2000), which are widely distributed in tropical and subtropical African freshwaters. They are small fishes predominantly found in riverine systems where they are mostly active at dusk. Several morphological synapomorphies support the monophyly of Petrocephalus (Taverne 1969, Taverne 1972, as do molecular data (Sullivan et al. 2000).
Central African Petrocephalus are particularly diverse and abundant throughout the large Congo basin with 18 species (Lavoué et al. 2010, Lavoué 2012. Here, we first describe a new species of Petrocephalus from the main channel of the Congo River at Yangambi, Central Congo basin. Based on our long-term work on Petrocephalus, we believe this new species is rare, as we have identified only one specimen after examination of hundreds of Central African Petrocephalus specimens. From the same locality, we examined two specimens of Petrocephalus that share more similarities to the specimens earlier identified as Petrocephalus binotatus from Odzala-Kokua (Lavoué et al. 2010) than to the holotype of P. binotatus, despite the greater proximity of Yangambi to the type locality of P. binotatus (Ikengo), than to Odzala-Kokua (Fig. 1). This led us to reevaluate this identification and describe the Odzala-Kokua and Yangambi specimens as new.

Materials and methods
Petrocephalus specimens newly examined in this study were collected in September 2010 from the Congo River at Yangambi, Orientale Province, Democratic Republic of Congo (Fig. 1). Comparative material including type specimens from all valid species from the Congo River basin and the Lower Guinea province is listed in Lavoué et al. (2004Lavoué et al. ( , 2010 and Lavoué (2012). About half of the nucleotidic sequences of the cytochrome b gene (about 600 base pairs from the 3' end) were determined for three specimens of Petrocephalus collected at Yangambi.
In the field, we recorded EODs from two of these three specimens of Petrocephalus. Each individual was first transferred to a plastic tank (ca. 80 cm long*30 cm wide*30 cm deep) filled with water from the capture location. EODs were recorded with silver/silverchloride electrodes connected to an Edirol FA-66 analog-to-digital Firewire audio interface (Roland Corporation). Signals were visualized and stored in SignalScope 2.0 virtual oscilloscope software (Faber Acoustical) such that positivity at the fish's head was always recorded in the upward direction. Water temperature of each recording was noted. Fish were euthanized with an overdose of MS222, photographed, tissued, tagged with a unique specimen number, fixed in buffered 10% formalin and later transferred to 70% ethanol.
Total peak-to-peak amplitudes of all EODs were normalized to a value of one. We did not correct the time base of EODs to a uniform temperature as this procedure pro-duces only minimal changes. Seven EOD measures were taken as described in Lavoué et al. (2008): relative height of peak 1, relative height of peak 2, relative height of peak 3 (when present), duration of peak 1, duration of peak 2, duration of peak 3 (when present), total EOD duration. EOD recordings have been archived in the Macaulay Library at the Cornell Lab of Ornithology (CUML accession numbers provided in Suppl. material 1).
Mature male specimens were identified by the presence of an anal fin notch (Pezzanite and Moller 1998). Methods for making counts and measurements and their abbreviations follow those given by Boden et al. (1997), except for body height and standard length that are modified as in Lavoué et al. (2004). Furthermore, the snout length and the mouth position are the nearest distances, parallel to the body axis, from the perpendicular line through the tip of the snout to the anterior border of the eye and the corner of the mouth, respectively.
We examined the specimens for the presence of each of the three distinct clusters of Knollenorgan-type electroreceptors on the head of Petrocephalus, as described in Harder (1968Harder ( , 2000. The "Augenrosettes" are directly above the anterior half of the eye, the "Nackenrosettes" are dorsally situated on each side of the nape, slightly anterior to the opercular openings and the "Kehlrosettes" are situated anterior to and extending somewhat below the insertion of the pectoral fins. DNA was extracted from fin clips preserved in 90% ethanol. PCR amplification and sequencing of the partial cytochrome b gene were as described by Sullivan et al. (2000) using the following Petrocephalus-specific primers: L15213_MOR (5'-CTA ACC CGA TTC TTT GCC TTC CAC TTC CT-3') and H15913_MOR (5'-TCG ATC TCC GGA TTA CAA GAC CG-3'). Cytochrome b sequences generated in this study are available in GenBank under accession numbers KF181719 to KF181721. The three new partial cytochrome b sequences were added to the dataset published in Lavoué (2012) from which individuals uninformative for interspecific comparisons have been removed. Following the conclusions of Kramer et al. (2012) who resurrected several species previously synonymized with Petrocephalus catostoma, we assigned revised names to two specimens (and their corresponding cytochrome b sequences) treated in Lavoué (2012): one specimen of P. catostoma from the Rufiji River is renamed Petrocephalus steindachneri and one specimen of P. catostoma from the Wami River is renamed Petrocephalus stuhlmanni. The alignment does not require any indels and missing data were coded with "-". The final alignment comprises 1140 nucleotidic positions. The maximum likelihood phylogenetic tree was calculated under the general time reversible model of nucleotide substitution with rate heterogeneity following a discrete gamma distribution (GTR+Г), using the software RAxML-VI-HPC (Stamatakis 2006) and its graphical interface raxmlGUI (Silvestro and Michalak 2012).
Description. Morphometric ratios and meristic data for holotype presented in Table 1. Standard length = 56.9 mm. Sex undetermined: specimen lacks anal fin "notch" present in mature males, but may be juvenile. Body ovoid, 2.7 times longer than high and laterally compressed. Head length 3.3 times in standard length. Snout short (HL/ SNL = 6.6) and round. Mouth small (HL/MW = 4.4), subterminal, opening under anterior half of eye. Teeth small and bicuspid, 13 in single row in upper jaw, 24 in single row in lower jaw. Nostrils closely set (distance between nostrils = 0.4 mm) with the posterior one very close to eye (distance between posterior nostril and eye = 0.1 mm). Dorsal and anal fins originating in posterior half of body (SL/PDD = 1.6 and SL/PAD = 1.8). Predorsal distance slightly greater than pre-anal distance (PDD/PAD = 1.1). Dorsal fin with 23 branched rays. Anal fin with 34 branched rays. Pectoral fins with 9 rays. Pelvic fin with 6 rays. Distance between pelvic fin and anal fin = 10.1 mm. Distance between pectoral fin and anal fin = 16.4 mm. Scales cover body, except for head. Lateral line visible and complete with 34 pored scales along its length. Ten scales around caudal peduncle. Ten scales between anterior base of anal fin and lateral line. Caudal peduncle narrow (CPL/CPD = 1.6). Thick skin on head. Knollenorgans visible, clustered into two distinct rosettes, Nackenrosette and Kehlrosettes as described in Harder (1968). Knollenorgan pores in front of eye not highly condensed in the form of a discernable Augenrosette.
Live coloration (Fig. 3). Body background color uniformly gray/silver with metallic reflection on the flanks and head. Light melanophores densely and evenly distributed on body, slightly larger on head. Three distinct black marks on each side of the body, one at the base of the pectoral fins, one at the base of the caudal fin and one subdorsal, below the first anterior rays of the dorsal fin. All fins hyaline.
Preserved coloration (Fig. 3). Background beige with dorsum slightly darker. Numerous light melanophores visible all over body. Thick skin on head and fins opaque. Three distinct melanin marks on side of body distinctly visible. Distribution (Fig. 1). Holotype (and only specimen known) of Petrocephalus boboto sp. n. collected from the main channel of the Congo River, at Yangambi, Orientale Province, Democratic Republic of Congo.
Phylogenetic affinities (Fig. 4). Petrocephalus boboto sp. n. represents a deep lineage within the genus Petrocephalus not closely related to any of the four described species exhibiting a similar pattern of markings: Petrocephalus zakoni, Petrocephalus odzalaensis, Petrocephalus balayi and Petrocephalus arnegardi sp. n. However, it is worth noting that Petrocephalus binotatus, the fifth species with a potentially similar pigmentation, is not included in this tree and the phylogenetic position of this species is unresolved.
Etymology. The name boboto is a word in Lingala, the language spoken at the type locality of Petrocephalus boboto sp. n., meaning "peace," alluding to the right of all people of D.R. Congo to live in peace and safety.
Comparison. The distinctive pattern of melanin markings of Petrocephalus boboto sp. n., having three dark spots-one at the origin of the pectoral fin, a second at the origin of the caudal fin, and a third on the flank just below the anterior base of the dorsal fin-distinguishes this species from most of its congeners. Only five other species of Petrocephalus similarly possess three black marks in these locations: P. odzalaensis, P. balayi, P. zakoni, P. binotatus and P. arnegardi sp. n. (the comparison with P. arnegardi sp. n. is provided under the description of this species). Petrocephalus boboto sp. n. can immediately be distinguished from P. zakoni by the presence of two of the three electroreceptor rosettes on the head that are typical to Petrocephalus (versus absence of all three in P. zakoni) and a higher number of anal fin rays (33 in P. boboto sp. n. versus   (CUMV 88076, 88079, 88041, 87838, 88063, 87785, 88052, 88053, 92390, 87830, 88080, 88123, 88064, 88065, 88031 and 88032) of Petrocephalus arnegardi sp. n., all from Odzala-Kokua National Park, and two other specimens (BMNH 2013.8.29.34 and2013.8.29.125) of Petrocephalus arnegardi sp. n. from Yangambi. Abbreviations: u= sex undetermined; m= sexually mature male; Min-Max= minimum-maximum; stdev= standard deviation. "*" indicates data from Pellegrin (1924  a maximum of 28 in P. zakoni). Petrocephalus boboto sp. n. can be distinguished from P. balayi and P. odzalaensis by its higher number of anal fin rays (33 in P. boboto sp. n. versus a maximum of 27 and 20 in P. balayi and P. odzalaensis, respectively). Petrocephalus balayi also has a proportionally larger mouth (HL/MW = 2.7-3.9, mean= 3.3 versus 4.4 in P. boboto sp. n.). Petrocephalus odzalaensis has a proportionally wider head (HL/IOW = 2.5-3.0, mean = 2.8 and HL/HW = 1.7-1.8, mean = 1.7 versus 3.7 and 2.7 in P. boboto sp. n., respectively) and its body background color is more pinkish rather than gray/silver in P. boboto sp. n. Petrocephalus boboto sp. n. is distinguished from the holotype of P. binotatus by a smaller mouth (SL/MW = 14.2 and HL/MW = 4.4 versus 11.6 and 3.2, respectively, in the holotype of P. binotatus), a smaller eye (HL/ED = 4.4 versus 3.6 in the holotype of P. binotatus), and a smaller interorbital distance (HL/IOW = 3.7 versus 2.3 in the holotype of P. binotatus).
The holotype of P. boboto sp. n. is smaller than the holotype of P. binotatus. The differences between P. boboto sp. n. and P. binotatus are unlikely to be the result of growth allometry since allometric differences have only been observed with respect to the number of teeth in some species of Petrocephalus (Bigorne and Paugy 1991), a character that does not differentiate these two species. Although we are confident of the specific distinction between P. boboto sp. n. and P. binotatus, more specimens are needed to better characterize the morphological, molecular and electrophysiological differences between them.
Electric organ discharge. Statistics for waveform landmarks and other EOD measurements are provided by Lavoué et al. (2008) for specimens recorded in Odzala-Kokua National Park, including the holotype and paratypes (specimens listed in Suppl. material 1). Petrocephalus arnegardi sp. n. produces EOD waveforms largely similar to those of many species of this genus. In Odzala-Kokua, mean EOD duration (± std. dev.) is 0.330 ± 0.074 msec in sexually mature males and 0.270 ± 0.033 msec in other sex undetermined specimens. The EOD waveform characteristics of the only recorded specimen of P. arnegardi sp. n. of Yangambi ( Fig. 2B; EOD biphasic, relative height of peak 1 = 0.216, relative height of peak 2 = -0.784, duration of peak 1 = 0.185 msec, duration of peak 2 = 0.075 msec, total EOD duration = 0.260 msec) are similar to those of Odzala-Kokua specimens in all respects. Based on characteristics of the EODs, the electrocytes are assumed to have non-penetrating stalks and to be innervated posteriorly (Sullivan et al. 2000).
Live coloration (Fig. 5; see also Fig. 3 in Lavoué et al. 2010). Body uniformly silvery white, with three distinct bilateral melanin marks: a distinct, ovoid black mark situated slightly anterior to the dorsal fin, sometimes covering only a few scales, a black spot at the base of the pectoral fin and a somewhat vertically oriented ovoid black mark centered at the base of the caudal fin that does not extend onto the upper and lower parts of the caudal fin. Fins hyaline.
Preserved coloration (Fig. 5; see also Fig. 3 in Lavoué et al. 2010). Body light brown, with head and dorsum slightly darker. Thick skin on head opaque. Numerous light melanophores on body, slightly larger ventrally from the snout to the anal fin. Fins whitish/opaque. Distribution (Fig. 1). Endemic to the Congo River basin. Holotype and paratypes from Odzala-Kokua National Park (Republic of the Congo) where they were mainly collected along the main channel of the Lékoli River, northwestern Congo River basin. The two specimens collected at Yangambi will extend the distribution to the eastern part of the Congo basin's central cuvette. Abundant in Odzala-Kokua but apparently rare at Yangambi (Poll and Gosse 1963).
Phylogenetic affinities (Fig. 4). The Yangambi specimens and the Odzala-Kokua specimens of P. arnegardi sp. n. are sister groups in the cytochrome b phylogenetic tree. Petrocephalus arnegardi sp. n. belongs to a clade containing P. odzalaensis and P. balayi, two other species with a similar melanin pattern composed of three distinct black marks, but it is only distantly related to P. boboto sp. n. As previously noted, P. binotatus is absent in this tree and its phylogenetic position is unknown.
Etymology. This species is dedicated to Matthew E. Arnegard, our friend and colleague, in recognition of his contributions to study of mormyrid evolution and diversification (e.g., Arnegard et al. 2005;Arnegard and Carlson 2005;Arnegard et al. 2010a;Arnegard et al. 2010b). Matthew Arnegard is additionally a member of the "Mintotom Team": researchers associated with the Carl D. Hopkins Laboratory at Cornell University who have conducted field studies on African weakly electric fishes for more than 15 years. ("Mintotom" is the plural form of the word for mormyrid fish in the Fang language of West Central Africa.).
Comparisons. As for P. boboto sp. n., the presence of three dark spots in P. arnegardi sp. n. distinguishes this species from most of its congeners. As for other species having a similar pattern of melanin marking, P. arnegardi sp. n. can easily be distinguished from P. zakoni by the presence of three electroreceptor rosettes on the head (versus absence of all three in P. zakoni) and a higher number of anal fin rays (a minimum of 30 in P. arnegardi sp. n. versus a maximum of 28 in P. zakoni). Its high number of anal fin rays distinguishes P. arnegardi sp. n. from P. balayi and P. odzalaensis (30-34 in P. arnegardi sp. n. versus a maximum of 27 and 20 in P. balayi and P. odzalaensis, respectively). Petrocephalus balayi has a proportionally larger mouth (HL/MW = 2.7-3.9 versus 4.1-5.0 in P. arnegardi sp. n.). Petrocephalus arnegardi sp. n. generally resembles to the holotype of P. binotatus, leading Lavoué et al. (2010) to assign these specimens from the Odzala-Kokua National Park to P. binotatus. However, Petrocephalus arnegardi sp. n. has a distinctly smaller mouth than P. binotatus  in Odzala-Kokua specimens and 4.2 and 4.5 in the two Yangambi specimens versus 3.2 in the holotype of P. binotatus) and a smaller interorbital width (HL/IOW ≥ 2.6 in Odzala-Kokua and Yangambi specimens versus 2.3 in the holotype of P. binotatus). The faded pigmentation in the pre- served holotype of P. binotatus precludes its accurate description and comparison (Fig.  6). Whereas a faded roundish black mark situated slightly anterior to the dorsal fin on each side of the flank and an ovoid black mark centered at the base of the caudal fin are visible on the preserved holotype, the presence of a black mark at the base of the pectoral fin is ambiguous (Fig. 6). In his description of P. binotatus, Pellegrin (1924) mentioned the subdorsal mark and the mark at the base of the anal fin, but did not make reference to any black mark at the origin of the pectoral fin. The black mark at the base of the pectoral fin in P. arnegardi sp. n. is always present and intense. Pellegrin also described the black mark at the base of the caudal fin as crescent-like, extending onto the upper and lower rays of this fin (see drawing in Pellegrin 1928) whereas on the holotype this mark appears more ovoid and does not seem to extend onto any fin rays (Fig. 6). Petrocephalus arnegardi sp. n. is distinguished from P. boboto sp. n. by a distinctly smaller mouth (SL/ MW ≥ 14.7, range = 14.7-18.4 versus 14.2 in P. boboto sp. n.), a slightly larger interorbital distance (HL/IOW ≤ 3.9, mean = 3.2 versus 3.7 in P. boboto sp. n.) and the presence of a well-defined Augenrosette (versus reduced in P. boboto sp. n.). In our phylogenetic tree (Fig. 4), P. boboto sp. n. is not the sister group of P. arnegardi sp. n. Eye relatively small (HL/ED ≥ 4.0); mouth subterminal (HL/MP ≥ 4.4), opening under the anterior half of the eye; two distinct melanin marks present but sometimes pale: a rounded sub-dorsal mark and a crescent-like mark at the base of the caudal fin; Knollenorgan electroreceptors organized into three distinct rosettes on head, but rosettes relatively small; EOD waveform typical for the genus, polarity normal (see Fig. 10C  Dorsal fin contains 24-26 branched rays; eye large (HL/ED ≤ 3.2); mouth very small relative to many congeners (HL/MW ≥ 5.2); two melanin marks present and distinct but of medium intensity: a rounded sub-dorsal mark and a crescent-like mark at the base of the caudal fin; two readily observable rosettes of Knollenorgan electroreceptors present on the head (Augenrosette and Nackenrosette) plus a Kehlrosette that is rather difficult to observe without staining .