225 Ichthyol. Explor. Freshwaters, Vol. 24, No. 3, pp. 225-236, 3 figs., 2 tabs., March 2014 © 2014 by Verlag Dr. Friedrich Pfeil, München, Germany – ISSN 0936-9902 Species limits and DNA barcodes in Nematolebias, a genus of seasonal killifishes threatened with extinction from the Atlantic Forest of south-eastern Brazil, with description of a new species (Teleostei: Rivulidae) Wilson J. E. M. Costa*, Pedro F. Amorim* and Giulia N. Aranha* Nematolebias, a genus of killifishes uniquely living in temporary pools of south-eastern Brazil, contains two nominal species, N. whitei, a popular aquarium fish, and N. papilliferus, both threatened with extinction and pres- ently distinguishable by male colour patterns. Species limits previously established on the basis of morphological characters were tested using mt-DNA sequences comprising fragments of the mitochondrial genes cytochrome b and cytochrome c oxidase I, taken from 23 specimens representing six populations along the whole geograph- ical distribution of the genus. The analysis supports the recognition of a third species, N. catimbau, new species, from the Saquarema lagoon basin, as an exclusive lineage sister to N. papilliferus, from the Maricá lagoon basin, and N. whitei, from the area encompassing the Araruama lagoon and lower São João river basins, as a basal line- age. The new species is distinguished from congeners by the colour pattern and the relative position of pelvic-fin base, besides 11 unique nucleotide substitutions. The distribution pattern derived from sister taxa inhabiting the Saquarema and Maricá basins is corroborated by a clade of the seasonal genus Notho lebias, suggesting a common biogeographical history for the two genera. Introduction concentrates a high number of species threatened with extinction (Tabarelli et al., 2005), many of Possibly the greatest present challenge for taxo- them still poorly known. Aplocheiloid killifishes nomists is to catalogue the poorly known species of the Neotropical family Rivulidae are particu- diversity of tropical areas under intense process larly diversified in the Atlantic Forest, where they of environmental degradation (Brook et al., 2006; are represented by eight endemic genera and Costa et al., 2012). For example, the Atlantic For- more than 40 endemic species (Costa, 2008, 2009, est of eastern Brazil, the second largest forest of 2010). Most killifishes endemic to this biome are South America and one of the richest biodiver- seasonal organisms, uniquely living in shallow sity centres in the world (Myers et al., 2000), temporary pools formed during the rainy seasons * Laboratório de Sistemática e Evolução de Peixes Teleósteos, Departamento de Zoologia, Universidade Fede- ral do Rio de Janeiro, Caixa Postal 68049, CEP 21944-970, Rio de Janeiro, RJ, Brasil. E-mail: [email protected] Ichthyol. Explor. Freshwaters, Vol. 24, No. 3 226 12 11 São João river basin 10 8 9 7 3 6 4 5 2 1 Saquarema system Araruama system Maricá system N 0 20 km 23°00'S 42°30'W 42°00'W Fig. 1. Geographical distribution of Nematolebias: , N. whitei; , N. papilliferus; , N. catimbau. Numbers indi- cate populations used in the analyses: 1, Inoã; 2, Maricá; 3, Sampaio Correia; 4, Bonsucesso; 5, São Pedro da Aldeia; 6, Caravelas; 7, Botafogo; 8, Barra de São João. (Myers, 1942; Costa, 2002a, 2009), besides being basin and the Araruama lagoon basin, and N. pa- restricted to small geographical areas and stand- pilliferus Costa, 2002, from two populations from ing among the most endangered vertebrates of the Maricá lagoon basin, and a single population South America (Costa, 2002b, 2012). from the Saquarema lagoon basin (Fig. 1). Both Nematolebias Costa, 1998 is a genus of sea- species were distinguished by characters of male sonal killifishes endemic to the Atlantic Forest of colour patterns, including the presence of golden the coastal plains of Rio de Janeiro state, south- lines on the dorsal fin which is present in N. papilli- eastern Brazil (Costa, 2002a). This region for- ferus but absent in N. whitei. More recent field merly comprised dense rain forests and broad work has revealed that the populations of N. papil- swampy areas (Wied-Neuwied, 1820), but since liferus from the Maricá basin, including the type the beginning of the 20th century it has been locality of the species, exhibit colour pattern mainly occupied by open vegetation formations slightly distinct from populations inhabiting the used as pasture for cattle, and more recently by Saquarema basin, making clear the necessity of a quick expansion of coastal urban centres. As a adding more data to test species limits. Thus, the consequence of habitat loss, endemic killifish objective of the present study is to combine revised species are severely threatened with extinction data of morphology at the population level with (Costa, 2009, 2012). mitochondrial DNA sequences obtained from six Nematolebias has been considered the sister populations representing the whole geographic group to a speciose clade containing all other taxa range of the genus. of the tribe Cynolebiasini, easily diagnosed by the presence of hypertrophied papillae on the pectoral fin in males and the presence of a broad Material and methods sub-distal orange stripe with overlapped golden lines on the anal fin in males (Costa, 2002a, 2006, Morphology. Data on colour patterns were pri- 2010). Costa (2002a) revised Nematolebias, recog- marily based both on direct examination of live nising two cryptic species (sensu Bickford et al., specimens during collections, and photographs 2007), N. whitei (Myers, 1942), a popular aquarium of both sides of live individuals, at least five males fish and known from some populations in a long and two females for each population, taken in geographical area between the São João river aquaria between about 4 and 12 hours after col- Costa et al.: New Nematolebias from south-eastern Brazil 227 lection, between 1994 and 2012. Other morpho- quencing reactions were performed in 10 μl reac- logical characters used in species description were tion volumes containing 1 μl BigDye 2.5, 1.55 μl obtained from specimens fixed in formalin just 5 × sequencing buffer (Applied Biosystems), 2 μl after collection, for a period of 10 days, and then of the amplified products (10-40 ng), and 2 μl transferred to 70 % ethanol. Material is deposited primer. The thermocycling profile was: (1) 35 in the following institutions: BMNH, Natural cycles of 10 seconds at 96 °C, 5 seconds at 54 °C History Museum, London; UFRJ, Instituto de and 4 minutes at 60 °C. The sequencing reactions Biologia, Universidade Federal do Rio de Janeiro; were purified and denatured and the samples and ZFMK, Zoologisches Forschungsmuseum were run on an ABI 3130 Genetic Analyzer. Se- und Museum Alexander Koenig, Bonn. Morpho- quences were edited using MEGA 5 (Tamura et metric and meristic data were taken following al., 2011) and subsequently adjusted manually Costa (1995); measurements are presented as (total of 1130 bp). percent of standard length (SL), except for those related to head morphology, which are expressed Species concept, species delimitation and diag- as percent of head length. Fin-ray counts include noses. The unified species concept (de Queiroz, all elements. Number of vertebrae and gill-rakers 2007) is herein adopted by expressing the con- were recorded from cleared and stained speci- ceptual definition shared by all traditional species mens (c&s) prepared according to Taylor & Van concepts (i. e. species are lineages united through Dyke (1985). Terminology for frontal squamation gene flow) when operational criterion elements follows Hoedeman (1958) and for cephalic neu- to delimit taxa are excluded from concepts. Any romast series Costa (2001). of those criteria may separately provide evidence about species limits independently from the DNA extraction, amplification and sequencing. other criteria (de Queiroz, 2007), but evidence Specimens were fixed in absolute ethanol im- extracted from multiple operational criteria is mediately after collection and later preserved in considered to produce stronger hypotheses of the same solution; see Appendix 1 for list of lineage separation (de Queiroz, 2007), thus con- specimens and respective GenBank accession gruent to the proposal for an integrative taxono- numbers. Total genomic DNA was extracted from my (Goldstein & DeSalle, 2010; Padial et al., 2010). muscle tissue of the caudal peduncle using the Species are herein recognised when their limits DNeasy Blood & Tissue Kit (Qiagen), following are concordantly supported by three different manufacturer instructions. To amplify the frag- operational criteria, two character-based and one ments of the mitochondrial DNA were used the tree-based (sensu Baum & Donoghue, 1995; Sites primers Cox1F and COIrev (Costa & Amorim, & Marshall, 2004), as described below. 2011; Sonnenberg et al., 2007) for the mitochon- The first method to delimit species used in drial gene cytochrome c oxidase I (cox1) and this study was the Population Aggregation primers L14724 and H15149 (Kocher et al., 1989; Analysis (Davis & Nixon, 1992), a character-based Meyer et al., 1990), for the mitochondrial gene method (hereafter PPA, following Sites & Mar- cytochrome b (cytb). Polymerase chain reaction shall, 2003), in which species are delimited by (PCR) was performed in 15 μl reaction mixtures unique combination of morphological character containing 5 × Green GoTaq Reaction Buffer states occurring in one or more populations. The (Promega), 3.6 mM MgCl2, 1 μM of each primer, analysis focused in diagnostic character states 75 ng of total genomic DNA, 0.2 mM of each dNTP used by Costa (2002a), besides checking other and 1U of Taq polymerase. The thermocycling characters commonly employed in killifish Sys- profile was: (1) 1 cycle of 4-5 minutes at 94 °C; tematics (e. g., Costa, 2006). Populations were (2) 35 cycles of 1 minute at 92 °C, 1 minute at seasonal pools or groups of neighbouring pools 48-54 °C and 1 minute at 72 °C; and (3) 1 cycle geographically isolated from other pools. PPA of 4 minutes at 72 °C.