Zootaxa 3669 (4): 503–521 ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2013 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.3669.4.5 http://zoobank.org/urn:lsid:zoobank.org:pub:DC8922B5-F317-46EC-92E0-7AED66A0C714 New Calisto species from Cuba, with insights on the relationships of Cuban and Bahamian taxa (Lepidoptera, Nymphalidae, Satyrinae) RAYNER NÚÑEZ AGUILA1, PÁVEL F. MATOS- MARAVÍ2,3 & NIKLAS WAHLBERG2 1División de Colecciones Zoológicas y Sistemática, Instituto de Ecología y Sistemática, Carretera de Varona km 3.5, Capdevila, Boye- ros, Ciudad de La Habana, Cuba 2Laboratory of Genetics, Department of Biology, University of Turku, FI–20014 Turku, Finland 3School of Biological Sciences, University of South Bohemia and Institute of Entomology, Biology Center AS CR, Branisovska 31, 37005 Ceske Budejovice, Czech Republic Abstract Three new species and a new subspecies of Calisto Hübner are described from Cuba, Calisto torrei sp. n. Núñez, Calisto dissimulatum sp. n. Núñez, Calisto aquilum sp. n. Núñez, and Calisto aquilum occidentalis ssp. n. Núñez. The immature stages of C. torrei and C. dissimulatum are also described. Notes on the distribution and biology of the species are given. All Cuban and Bahamian taxa form a monophyletic group which seems to have originated in northeastern Cuba spreading later to the west. DNA sequence data also allowed to recognize both Bahamian taxa, Calisto sibylla and Calisto apollinis stat. n., as distinct species, and to synonymize Calisto herophile parsonsi syn. n. under Calisto herophile. Key words: West Indies, Bahamas, DNA, molecular systematics, genetic distance, taxonomy, immature stages, distribu- tion, checklist, speciation, synonymy Introduction The butterflies of the genus Calisto have evolved in isolation and radiated in the West Indies (Smith et al. 1994; Miller & Miller 2001; Lamas 2004; Sourakov & Zakharov 2011). Until recent times Hispaniola was considered to be the only island where actual diversification took place, from where about 30 native species are known today, with the remaining nearby islands populated by one or two species (Smith et al. 1994; Miller & Miller 2001; Sourakov & Zakharov 2011). The Herophile species group, sensu Bates (1935), is used to describe all Calisto found on Cuba and Bahamas. Brown & Heineman (1972) reduced this group to two species shared by the archipelagos, a change followed by the majority of subsequent workers (Smith et al. 1994; Miller & Miller 2001; Hernández 2004; Lamas 2004). An intense sampling effort during the last few years in Cuba revealed a very different situation. Núñez et al. (2012) proposed specific status for most of the Calisto taxa previously described from that island and named a new species based on morphological and molecular data. Núñez et al. (2012) also included the description of the immature stages of four Cuban species, known before only by an incomplete description of C. herophile by Dethier (1940) and Torre (1968). The results of Núñez et al. (2012) also indicated that more findings were on the way. The elevation of former subspecies to species and the presence of “orphan” lineages in the DNA-based phylogeny representing potential new taxa encouraged us to continue research on Cuba. In the present article, three new species of Calisto are described from Cuba, including the immature stages of two of them. We also provide a phylogenetic hypothesis of the relationships of Cuban and Bahamian species of Calisto. Finally, an updated checklist of all known Cuban and Bahamian species of the genus containing a full synonymy and all recent changes is given. Accepted by Carlos Prieto: 2 May 2013; published: 10 Jun. 2013 503 Materials and methods Procedures for dissecting and preparing genitalia and terms for wing pattern follows that of Núñez (2009), and Smith et al. (1994), Johnson and Hedges (1998) and respectively. For the female genitalia, terminology detailed by Johnson et al. (1987) was followed. Eggs were obtained by confining females to plastic jars of 5 oz. where the eggs remained until larvae hatched. Larvae were raised in Havana, Cuba, in May–August/2012, Calisto torrei sp. n., and April–May/2012, C. dissimulatum sp. n., at the ambient temperature, humidity and photoperiod. For all species, an introduced common lawn grass, Zoysia matrella (L.) was used daily as substitute host plant. Width and height of head capsules and length of the first instar, were measured with an ocular micrometer (precision = 0.01 mm mounted on a Carl Zeiss Stemi 2000 stereoscopic microscope. Last instar and pupae were measured with a metric ruler (precision = 1 mm). For the description of immature stages we used characters described by Sourakov and Emmel (1995) and by Sourakov (1996, 1999). We followed Dethier (1940) when describing the larval striation with the exception of the para–dorsal line, to which we referred as “subdorsal line” instead. Type material is deposited at the entomological collection of the Institute of Ecology and Systematics (CZACC), Havana, Cuba. Two butterfly legs per individual were preserved either by desiccation or in ethanol. DNA was extracted from legs using the DNEasy extraction kit (QIAGEN). Six molecular markers including one mitochondrial (COI) and five nuclear genes (EF1α, wingless, GAPDH, RpS5 and CAD) were amplified using previously published primers and protocols (Wahlberg and Wheat 2008). DNA sequencing was carried out by the company Macrogen-South Korea. Sequence editing and alignment were done manually in the program BioEdit v7.0.9 (Hall 1999). Voucher photos are available at the Nymphalidae Systematics Group database (http://www.nymphalidae.net/nsg_db/) and DNA sequences have been submitted to GenBank (see Appendix 1 for accession numbers). DNA voucher specimens are deposited at CZACC, Cuban material, and at the collection of the McGuire Center for Lepidoptera and Biodiversity, Gainsville, USA, in the case of the Bahamian one. Genetic distances within and among taxa were calculated using the program MEGA5.1 (Tamura et al. 2011) using the Pairwise Distance Calculation analysis for the partial sequence of COI gene, following the DNA barcoding approach (Hebert et al. 2003, Hebert et al. 2004). New sequences were added to the dataset of Núñez et al. (2012). Phylogenetic analyses of the combined molecular data were carried out in the program MrBayes v3.2 (Ronquist et al. 2012) and executed through the Bioportal web portal (https://www.bioportal.uio.no). The data were partitioned by gene and analyzed as independent partitions. Various possible models of molecular evolution were sampled for each partition during the analysis by taking advantage of the model-jumping feature of MrBayes v3.2 (Ronquist et al. 2012) through the command “lset applyto = (all) nucmodel=4by4 nst=mixed rates=gamma covarion=no;”. Two independent MCMC analyses with four simultaneous chains (one cold and three heated) for each analysis were run for 10 million generations and the sampling of trees and parameters was set to every 1000 generations. Convergence of the two runs was determined by the stationary distribution plot of the log likelihood values against number of generations and confirmed by the average standard deviation of split frequencies which in all the cases were lower than 0.05. We discarded the first 2,500,000 generations as burn-in and trees were summarized under the 50 percent majority rule method. Results Calisto torrei Núñez sp. n. Figures 1–4, 17–18, 23, 26, 29, 33, 34, 37–50 Type material. Holotype—♂, CUBA, Sancti Spiritus, Banao, ascent to (“subida a” in the original label) La Sabina, 450m, 10/V/2012, 21°52'48"N 79°35'32"W, R. Núñez, DNA voucher RN01–01 (M065). Deposited in CZACC. Paratypes—9 ♂, 7 ♀: same data as holotype (1 ♂); northern slope of (“ladera norte de” in the original label) Pico Potrerillo, 750–850m, 11/V/2012, 21°53'27"N 80°00'49"W, R. Núñez, DNA voucher RN01–02 (M066) (3 ♂); Topes de Collante, Mi Retiro, V/2002, 21°53'41"N 80°01'02"W, R. Núñez, DNA voucher PM15–01 504 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. (M047), genitalia ♂ & ♀ in glycerine, prep. legs RNA209/210, wings RNA166/199/241 (2 ♂, 2 ♀); ; Deposited in CZACC. Cienfuegos, Buenos Aires, 600m, 16/VI/1967, 21°59'13"N 80°11'20"W, prep. wings RNA272 (1 ♀); same data as preceding except V/2006, R. Núñez, DNA voucher PM07–11 (M018), genitalia in glycerine, prep. wings RNA197 (1 ♀); Pico San Juan, 1140m, V/2006, 21°59'25"N 80°08'50"W, R. Núñez (2 ♂); same data as preceding except 12/V/2012, R. Núñez, DNA voucher RN01–03 (M067) (1 ♂, 2♀); same data as preceding except ex ova, emerged 10/VIII/2012 (1 ♀). Deposited in CZACC. Etymology. The name honors Salvador Luis de La Torre who devoted his life to the study of Cuban Lepidoptera and described several native Calisto taxa. Diagnosis. Calisto torrei is superficially most similar to Calisto bradleyi Munroe 1950, Calisto muripetens Bates 1939 and Calisto occulta Núñez 2012. From the first, it differs by its larger average size: forewing length (FWL) ♂ (Mean ±SD) = 21.3±1.0 mm, N=10 ♀ FWL=23.2±1.0, N=6 versus ♂ FWL=19.3±0.8 mm, N=15, ♀ FWL=19.4±1.1 MM, N=15. Calisto torrei also differs by the presence of an apical rounded lobe on androconial patch and the lack of a small bar of iridescent blue scales at underside of anal lobe that is present in C. bradleyi. From C. muripetens and C. occulta, C.torrei differs by having the anterior margin of androconial patch located behind the posterior margin of cell instead ahead, so its overall shape is slender Calisto torrei has also a smaller area of the cell, about 33–40%, covered by red scales whereas in C.