A new species of inseminating seasonal killifish of the Cynopoecilus melanotaenia complex from southern Brazil (Cyprinodontiformes: Rivulidae)

Abstract Background The Cynopoecilus melanotaenia complex is a morphologically homogeneous killifish group, endemic from an area encompassing southern Brazil and northeastern Uruguay. It presently comprises four valid species: C. melanotaenia, the type species of the genus, and C. fulgens, C. intimus, and C. nigrovittatus. New information Cynopoecilus feltrini, n. sp., from the lower Tubarão river basin, southern Brazil, is distinguished from all congeners of the C. melanotaenia complex by having frontal E-scales medially overlapped, branchiostegal region orangish red in males and dorsum with few dark brown spots above opercular region. A phylogenetic tree derived from the analysis of a fragment of the mitochondrial gene cytochrome c oxidase subunit I (681 bp) indicates that C. feltrini is a member of the clade that includes all species of the C. melanotaenia complex except C. melanotaenia, as well as that C. feltrini is the sister group of a clade comprising C. fulgens and C. nigrovittatus.


Introduction
Killifishes of the tribe Cynopoecilini (Order Cyprinodontiformes, Family Rivulidae) comprise a well corroborated clade endemic of South America, between eastern Brazil and northeastern Uruguay (Costa 2008, Costa 2014. They are members of the subfamily Cynolebiasinae, which uniquely includes seasonal species living in temporary pools (e.g., Costa 2010). Two cynopoeciline genera, Campellolebias Vaz-Ferreira and Sierra 1974 and Cynopoecilus Regan 1912, are remarkable by containing the only internal fertilizing species of the suborder Aplocheiloidei (Vaz-Ferreira and Sierra 1974, Costa et al. 1989, Costa 1998.
Cynopoecilus is diagnosed, among other characters, by a morphological apparatus on the male anal fin related to insemination that is unique among killifishes (Costa 1995). It consists of a series of minute rays on the anterior part of the fin, forming an inseminating structure (Costa 1995, Costa 1998). In addition, Cynopoecilus may be easily diagnosed by the unique colour pattern occurring in all included species, consisting of a dark reddish brown to black stripe along the lateral midline of the body and another between the pectoral-fin base and the posterior end of the anal-fin base (Costa 1998, Costa 2002, Ferrer et al. 2014.
During the 90 years following its first description by Regan (1912), Cynopoecilus was considered as a monotypic subgenus of Cynolebias Steindachner 1876 (Myers 1952, Vaz-Ferreira and Sierra 1971, Parenti 1981, or as a monotypic genus (Costa 1990, Costa 1995, occurring in a vast area of southern Brazil and northeastern Uruguay (Vaz-Ferreira andSierra 1971, Costa 1995). After larger collections made between 1997 and 1999, Costa (2002) provided the first taxonomical revision of Cynopoecilus, recognising five species: C. melanotaenia (Regan 1912), the type species of the genus, and C. fulgens Costa 2002, C. intimus Costa 2002, C. multipapillatus Costa 2002, and C. nigrovittatus Costa 2002; but a unpublished taxonomic revision of the genus by the first author indicates that C. multipapillatus is a synonym of C. fulgens. These species form a morphologically homogeneous group, herein called the Cynopoecilus melanotaenia complex, with all included species highly differing from another congener recently described by Ferrer et al. (2014), C. notabilis Ferrer et al. 2014. Among several diagnostic characters, species of that complex have the dorsal-fin origin in a vertical just anterior to anal-fin origin in males (vs. anterior to pelvic-fin insertion in C. notabilis), caudal fin rounded to sub-truncate in males ( vs. lanceolate), unpaired fins hyaline in males, with pale spots or spots absent (vs. yellow with dark red spots), absence of a dark red stripe on the basal portion of the anal fin in males (vs. present), and flank in females without bars (vs. with bars).
More recently, collecting trips provided large new collections making possible a detailed revision of the C. melanotaenia complex, which is presently in progress. Among the new findings is a new species herein first described after evidence from morphology and mitochondrial DNA.

Materials and methods
Material is deposited in the ichthyological collection of the Institute of Biology, Universidade Federal do Rio de Janeiro, Rio de Janeiro (UFRJ). Specimens were euthanized just after collection in a buffered solution of ethyl-3-amino-benzoat-methansulfonat (MS-222) at a concentration of 250 mg/l, for a period of 10 minutes or more, until completely ceasing opercular movements. Specimens fixed in formalin just after euthanasia for a period of 10 days, and then transferred to 70 % ethanol; specimens used in the molecular analysis were fixed in 98 % ethanol just after euthanasia and later preserved in the same fixative. List of specimens and respective GenBank accession numbers appear in Table 1 Data on colour patterns were based numerous photographs of both sides of five live males and three live females, taken in aquaria between 1 and 78 hours after collection. Morphometric and meristic data were taken following Costa (1998); measurements are presented as percent of standard length (SL), except for those related to head morphology, which are expressed as percent of head length. Fin-ray counts include all elements. Number of vertebrae and gill-rakers were recorded from cleared and stained specimens; the compound caudal centrum was counted as a single element. Osteological preparations (c&s) were made according to Taylor and Dyke (1985). Terminology for frontal squamation follows Hoedeman (1958) and for cephalic neuromast series Costa (2001).
Total genomic DNA was extracted from muscle tissue of the right side of the caudal peduncle using the DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer instructions. To amplify the fragment of the DNA were used the primers L4299, H2198 (Folmer et al. 1994) and COX1F and COX1R (Costa and Amorim 2011), specific for the mitochondrial fragment of the cytochrome c oxidase subunit I (COX1). Polymerase chain reaction (PCR) was performed in 30μl reaction mixtures containing 5x Green GoTaq Reaction Buffer (Promega), 3.2 mM MgCl , 1 μM of each primer, 75 ng of total genomic DNA, 0.2 mM of each dNTP and 1U of Taq polymerase. The thermocycling profile was: (1) 1 cycle of 4 minutes at 94°C; (2) 35 cycles of 1 minute at 92°C, 1 minute at 50°C and 1 minute and 30 seconds at 72°C; and (3) 1 cycle of 4 minutes at 72°C. In all PCR reactions, negative controls without DNA were used to check contaminations. Amplified PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Sequencing reactions were made using the BigDye Terminator Cycle Sequencing Mix (Applied Biosystems). Cycle sequencing reactions were performed in 10 μl reaction volumes containing 1 μl BigDye 2.5, 1.55 μl 5x sequencing buffer (Applied Biosystems), 2 μl of the amplified products (10-40ng), and 2 μl primer. The thermocycling profile was: (1) 35 cycles of 10 seconds at 96°C, 5 seconds at 54°C and 4 minutes at 60°C. The sequencing reactions were purified and denatured and the samples were run on an ABI 3130 Genetic Analyzer. Sequences were edited using MEGA 6 (Tamura et al. 2013) and aligned using ClustalW (Chenna et al. 2003).
Species were diagnosed using two criteria: unique combination of morphological character states (diagnosability criterion; e.g., Davis and Nixon 1992) and DNA haplotype tree (e.g., Wiens and Penkrot 2002). In the molecular phylogenetic analysis, terminal taxa included all species of the C. melanotaenia complex, except C. intimus, which was not found in recent collections due to deep habitat loss in the type locality area; outgroups were C. notabilis, the sister group of the C. melanotaenia complex; Campellolebias brucei Vaz-Ferreira and Sierra 1974, the type species of Campellolebias, the sister group of Cynopoecilus; and Notholebias fractifasciatus (Costa 1988), a member of Notholebias, a basal genus of the Cynopoecilini. Sequences were aligned using Clustal W (Chenna et al. 2003), after which the DNA sequences were translated into amino acids residues with MEGA 6.0 to test for the absence of premature stop codons or indels. The dataset was partitioned according to each codon position. The best fitting evolutionary model of each partition using Akaike information criteria (AIC) was determined with the software jModeltest version 2.  median opercular 1, ventral opercular 2-3, mandibular 8-10, lateral mandibular 7, paramandibular 1. Gill-rakers on first branchial arch 2 + 9. Six branchiostegal rays.
Dorsal and anal fins pointed in males, rounded in females; caudal fin rounded in both sexes; often short filamentous ray on tip of dorsal and anal fins, and minute posterior filamentous extension on middle of caudal fin in males. No scales on dorsal and anal fins, scales extending on about 30 % of caudal fin. Four to six neuromasts on caudal-fin base. Pectoral fin rounded, posterior margin reaching vertical between anus and urogenital opening in males, shorter, not reaching pelvic-fin base in females. No scales on pectoral-fin base. Pelvic-fin small, tip reaching between anus and urogenital opening in males, not reaching anus in females; pelvic-fin bases medially in close proximity. Dorsal-fin origin in vertical just anterior to anal-fin origin. Dorsal-fin origin between neural spines of vertebrae 12 and 13; anal-fin origin between pleural ribs of vertebrae 10 and 12 in males, between pleural ribs of vertebrae 12 and 13 in females. Hypurals forming single plate. Ventral process of posttemporal absent. Dorsal-fin rays 18-19; anal-fin rays 25-27; caudal-fin rays 29-32; pectoral-fin rays 13-15; pelvic-fin rays 5-7. No contact organ on fins.
Colouration. Males: Side of body pale brown to light yellowish brown, lighter on ventral portion; broad dark reddish brown to black stripe between posterior orbital margin and caudal-fin base, other similar narrower stripe between pectoral-fin base and posterior portion of anal-fin base; longitudinal rows of greenish blue to greenish golden spots, consisting of one spot per scale, on head side and flank, sometimes interrupted or rudimentary. Dorsum pale brown, with few dark brown spots above opercular region. Ventral portion of head and venter white. Lower jaw dark reddish brown. Few dark reddish brown on suborbital region. Branchiostegal membrane orangish red. Iris yellow, with dark reddish brown bar on middle, anterior and posterior portion with greenish Cynopoecilus feltrini holotype dorsal view of the head, white circles highlight E-scales borders overlapped. golden iridescence. Unpaired fins pale grey; small dark grey spots on basal region of dorsal fin; light blue iridescence on margins of caudal and anal fins. Pectoral fin hyaline. Pelvic fin pale grey.
Females: Colour pattern similar to that described for males, but iridescent colour paler, median stripe often forming row of dark brown or black blotches, and faint grey dots present on basal portion of anal fin.

Diagnosis
Distinguished from all other species of the Cynopoecilus melanotaenia complex by having frontal E-scales medially overlapped (vs. separated by interspace), branchiostegal region orangish red in males (vs. hyaline to pinkish hyaline), dorsum with few dark brown spots above opercular region (vs. dark brown spots over most region between snout and dorsal-fin origin).

Etymology
Named after Caio Feltrin, in recognition of his dedication in inventorying the fish fauna of southern Brazil.

Distribution
Known only from the type locality area, in temporary pools in the floodplains of the Tubarão river, Santa Catarina state, southern Brazil, corresponding to the northernmost record for the genus Cynopoecilus (Fig. 3). Distribution of Cynopoecilus species. Source: Costa (2002), Ferrer et al. (2014) and the current study for C. feltrini.

Taxon discussion
Cynopoecilus feltrini is easily identifiable by some morphological characters. Among them, the frontal squamation pattern consisting of E-scales medially overlapped is unique among cynopoeciline killifishes, which have been diagnosed by the E-scales medially separated by an interspace (Costa 1998). Another striking feature of C. feltrini is the orangish red branchiostegal region in males, a condition not found in other congeners of the C. melanotaenia complex, but similar to the red branchiostegal region of C. notabilis (Ferrer et al. 2014). Finally, all species of the C. melanotaenia complex may be easily recognised among other cynopoecilines by the presence of numerous dark brown spots along the dorsum, but in C. feltrini these spots are restricted to region above the opercular region.
Cynopoecilus feltrini is presently known only from the lower Tubarão river basin, which is the northern-most record of the genus. The phylogenetic tree supports C. feltrini as a member of the clade that includes all species of the C. melanotaenia complex except C. melanotaenia, which is the taxon endemic of an area corresponding to the southern- Maximum parsimony tree obtained from the COXI sequences. Numbers below the nodes are referring to the value of Bootstrap test and over the nodes are the posterior probability of the Bayesian inference analysis, asterisks mean value of 100. Sequences used to generate the trees are listed in Table 1. Adjacent color bar indicates the species distribution area within shown in Fig. 3. most region of the genus distribution (Figs 3, 4). The analysis also supports sistergroup relationships between C. feltrini and a clade comprising C. fulgens, from the coastal plains between the Patos lagoon and the sea, and C. nigrovittatus, from the lower Jacuí river drainage (Figs 3, 4). In addition, all the lineages corresponding to the four species of the C. melanotaenia complex sampled in this study had high bootstrap values in the analysis, this corroborating morphological data (Costa 2002, the present study). Cynopoecilus intimus, endemic of the upper Jacuí river basin, still has its phylogenetic position undetermined, since it was the only species of the C. melanotaenia complex not sampled for molecular data and morphological data alone do not provide unambiguous evidence about its position. In addition to diagnostic characters distinguishing C. feltrini from all other congeners discussed above, C. feltrini is easily distinguished from C. intimus by the latter having large dark brown blotches on the flank immediately below the dorsal-fin base (vs. blotches absent in C. feltrini) and having minute pelvic fin in males, pelvic-fin length 4.7-5.6 % SL, its tip reaching anus or shorter (vs. 5.9-7.3 % SL in C. feltrini, pelvic-fin tip reaching space between anus and urogenital opening).

Notes
This taxon was included as terminal in phylogenetic analysis of this study. Genbank access code to the sequences in Table 1