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 , with insights on the relationships of Cuban and Bahamian taxa (, , )

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 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 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. muripetens and C. occulta this area is 50–66%. Internally, C. bradleyi female genitalia of is proportionally smaller and its dorsal crown is very thin compared to that of C. torrei. Description. Forewing length ♂ 19.7–23.0 mm, ♀ 21.2–24.3 mm. Dorsal surface of wings brown, uniform in both sexes; male androconial patch darker than background, almost black, anterior margin behind posterior margin of cell, overall shape as an obtuse slender triangle due to oblique outer margin (Figures 1, 3, 23). Ventral wing surface brown with darker lines, background slightly paler distal of post discal line; forewing ocellus with two bluish white iridescent pupils, post discal line externally edged with pale yellow scaling at ocellus area; cell colored red at the middle half to two thirds, both base and apex brown-colored as surrounding background; hindwing background mixed with pale yellow and ochre scales basal to post discal line which is externally edge by pale yellow scaling, heavier around ocellus; post discal area splashed with pale yellow and lilac scales and three white dots at vein interspaces M1–M2, M2–M3 and M3–Cu1, central dot of row distinctly larger than others; both subterminal lines slightly rust-colored near apex (Figures 2, 4, 17, 18). Male genitalia with tegumen about two thirds the length of uncus, flat and rounded at posterior half; uncus slightly arched and gradually tapering toward apex; digitiform projection of valvae stout, slightly curved to straight at both margins; aedeagus sinuate with an almost indistinct left curve at basal and a pronounced right curve at middle of apical half (Figure 26). Female genitalia with dorsal crown tall, height about 0.4x width; corpus bursae about the same length of ductus bursae (Figure 29). Distribution. Calisto torrei has been collected in few localities on both sections of the Guamuhaya massif (Alturas de Banao and Alturas de Trinidad), the major mountain range of central Cuba (Figures 33, 34). Biology. Collecting sites are at low to moderate elevations (450–1140 m). Habitats include broad leaf evergreen forest, rainforest, and mogotes' (limestome hills) vegetation complex (Figures 37–40). Individuals have been observed taking nectar on Palicourea domingensis (Rubiaceae). On May 13th 2012 a captive female laid two pale yellow eggs glued to the walls of a plastic container. After 24 hours, egg color turned beige with tiny orange brown spots. A single larva hatched 7 days after oviposition and ate the entire corion. First instar (Figure 41)—Head width 0.72 mm, height 0.70 mm. Initial length 3.50 mm, final length 4.9 mm. Head dark brown, almost black; body pale beige with 7 thin reddish brown longitudinal lines. After feeding on grass, the background turned pale green. First molt occurred after 7days. Second to fifth instars (Figures 42–47) After first molt, the coloration of larva changed to a new pattern that persisted through subsequent instars with the only variation being a gradual darkening toward final instar. Head width, height, final length and duration of second to fifth instars were: 0.93, 0.98, 8.5 mm, and 7 days at the 2nd, l.30, 1.33, 12 mm, and 9 days at the 3rd, 1.85, 1.95, 17 mm, and 11 days at the 4th, and 2.58, 2.82 , 25 mm, and 15 days at the 5th, respectively. Head background pale grayish brown with the following dark brown, almost black, marks: lateral vertical lines from stemmata to horns continue to meet at middle of top of head; X shaped mark with upper arms short, almost indistinct on the epicranium; a transverse band across the frontoclypeus interrupted at the middle of frons. Body background stramineous with an increasing ochre tint toward final instar; dorsal line pale

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 505 brown with dots on sides at the middle of each segment; other lines only slightly darker than background and gradually fading toward last abdominal segment; subdorsal lines in zig zag with angles closest to dorsal line at the beginning and end of each segment; suprastigmatal line the thickest one, straight, darkened forming dots over spiracles; stigmatal and infrastigmatal lines thinner, convex between spiracles. After it stopped feeding, the mature larva remained straight for 2 days, emptied its digestive tract, wove a thin silk thread and hung head down, in J position, and pupated one day later. Pupa (Figures 48–50)—Length 12 mm, maximum width 4.8 mm. Background pale brownish gray, darker at sides of abdomen; a pair of ventral black dots on eyes and another pair at apex of metathoracic tibiae; diffuse orange coloration at the base of labial palpi and at sides of antennae tips; a row of tiny dark brown dots on veins near wing outer margin; wing sheaths with a darker diffuse spot at the middle and another darker near costa at apical third; abdomen with a dark brown line on sides, abdomen with dorsal transverse ridges reduced to a pairs of small crests on segments 1 to 6; last abdominal segment long, stout, cremaster area enlarged, broad and slightly flattened. The adult emerged in the afternoon, 3 to 4:00 pm, of August 10th after 11 days. Total developing time was 93 days. The natural host plant is unknown. Remarks. Calisto torrei represents one of the “orphan” lineages found in the molecular results of Núñez et al. (2012), where it was represented by the specimen coded as PM07-11. Following this lead, the new taxon was discovered after reviewing the available collections, the field collection of more individuals including immature stages, and the sequencing of more specimens. It was confused with sympatric Calisto muripetens Bates by Fontenla & Rodríguez (1990) and Núñez et al. (2012). DNA analysis showed genetic distances of 5.0 and 8.5% from sympatric C. muripetens and C. aquilum (a new species described below), respectively. Close relatives, C. bradleyi and C. occulta, are separated by 4.8% and 5.0% and have their populations more than 300 and 450 km away, respectively. Intraspecific variation was 0.15% for C. torrei (4 specimens, 4 localities), 0% for both C. bradleyi (3 specimens, 1 locality) and C. muripetens (3 specimens, 3 localities), and 0.82% for C. occulta (5 specimens, 2 localities). Immature stages also showed diagnostic features for C. torrei. Element patterns of larval head and body exhibit a unique configuration when compared with larvae of other Cuban Calisto including those of Calisto bruneri Michener 1949, C. muripetens and C. bradleyi that will be described in a future article. Similarly the pupa shows a characteristic grayish ochre background different from that of C. occulta, which is yellower, although it exhibits a comparable spot pattern.

Calisto dissimulatum Núñez, sp. n. Figures 5–8, 19–20, 24, 27, 30, 35, 51–67

Type material. Holotype—♂, CUBA, Holguín, (“Estación La Zoilita” in the original label) La Zoilita Station, 400 m20/II/2010, 20°37'42"N 75°29'08"W, R. Núñez, DNA voucher PM07–20 (M037). Deposited in CZACC. Paratypes—16 ♂, 9 ♀: same data as holotype except 3–10/IV/2012, DNA vouchers RN01–08 (M072) & RN02– 01 (M073), genitalia ♂ & ♀ in glycerine (6 ♂, 3 ♀); same data as preceding except ex ova, emerged 6–16/VI/2012 (8 ♂, 5 ♀); road to (“camino de” in the original label) La Zoilita, IX/1986, 20°38'N 75°29'W, R. Rodríguez (2 ♂, 1 ♀).Deposited in CZACC. Additional material. 9 ♂, 3 ♀: Santiago de Cuba, Baire, road to (“camino a” in the original label) Las Manuelas, 450m, 20°13'09"N 76°21'52"W, 23/IX/1952, S.L. de La Torre (1 ♂); Loma del Gato, 20°00'34"N 76°02'16"W, 6/IX/1951, S.L. de La Torre, genitalia ♂ in glycerine (4 ♂, 2 ♀); same data as preceding except 17– 20/VI/1952, F. de Zayas & P. Alayo, genitalia in glycerine, prep. wings RNA187, prep. legs RNA222 (3 ♂); in CZACC. Guantánamo, Piedra La Vela, 650m, VII/2001, 20°24'45"N 74°56'51"W, R. Núñez (1 ♂); Guantánamo, Piedra La Vela, El Mulo, 600m, VII/2001, 20°25'27"N 74°54'32"W, R. Núñez (1 ♂). Deposited in CZACC. Etymology. The species name is derived from the Latin dissimulo (dissimulate, feign), in reference to its great resemblance with Calisto brochei Torre 1973 and in a less extent with Calisto smintheus Bates 1935. Diagnosis. Calisto dissimulatum is superficially most similar to Calisto smintheus Bates 1935, Calisto brochei Torre 1973 and Calisto aquilum described below. From the first two, it differs by having an enlarged white dot at ♂ M2–M3, a lesser area covered by red scales on underside of forewing cell, and a smaller average size:

506 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. FWL=17.2±0.7 mm, N=16, ♀ FWL=18.6±0.3 mm, N=7 versus ♂ FWL=21.9±1.6 mm, N=50, ♀ FWL=22.5±1.4 mm, N=23 of C. smintheus and ♂ FWL=20.9±0.8 mm, N=9, ♀ FWL=21.5±0.4 mm, N=5 of C. brochei. From C. smintheus also differs by its less bright overall ventral wing pattern and the absence of the strong reddish splashing at basal two thirds at underside of hindwing. From C. brochei also lacks the waved outer margin of male androconial patch. Internally, the male genitalia of C. dissimulatum possess an uncus proportionally smaller in relation to tegument; the latter is tapered from the mid-point onwards, unlike C. smintheus and C. brochei, where it tapers starting from the base The digitiform projections of valvae are straight as in C. smintheus but shorter whereas they are slightly curved in C. brochei. Differences from C. aquilum will be treated below in the latter species diagnosis. Description. Forewing length ♂ 16.3–18.1 mm, ♀ 18.1–18.8 mm. Upper surface of wings dark brown, uniform in both sexes; male androconial patch darker than background, almost black, anterior margin behind posterior margin of cell, outer margin parallel to wing margin (Figures 5, 7, 24). Under surface brown with darker lines, background slightly paler at outer side of post discal line; forewing ocellus with two bluish white iridescent pupils, post discal line externally edged with pale yellow scaling at ocellus area; about two fifths of cell extension red colored, both base and apex brown colored as surrounding background; hindwing background mixed with pale yellow and ochre scales basal to pos dical line which is externally edge by dense pale yellow scaling, heavier around ocellus; post discal area splashed with pale yellow and lilac scales and four white dots at vein interspaces

Rs–M1, M1–M2, M2–M3 y M3–Cu1, dot at M2–M3 distinctly larger than M3–Cu1 one and slightly larger than remaining ones; apex of both wings densely covered by ochre scaling around subterminal lines (Figures 6, 8, 19, 20). Male genitalia with tegumen about two thirds the length of uncus, slightly concave at the middle and rounded at posterior half; uncus slightly arched, basal half about the same height then gradually tapering toward apex; digitiform projection of valvae, including ventral margin, straight and moderately short; aedeagus sinuated with distinct right curves at both basal and apical halves and an additional smaller curve at the middle (Figure 27). Female genitalia with dorsal crown tall, height about 0.33x width; corpus bursae about the same length of ductus bursae (Figure 30). Distribution. Calisto dissimulatum is known from few localities of the western halves of the Nipe–Sagua– Baracoa and Sierra Maestra, the two major mountain ranges of eastern Cuba (Figures 33, 35). Biology. Collecting sites are at low elevations (400–850 m). Habitats include rainforest, broad leaf evergreen forest, pine forest, and charrascales (scrub forest) (Figures 51, 52). On April 5th 2012 a female was observed at 12:30 pm ovipositing twice on a small grass species. A three egg cluster and one single egg were laid glued to the underside of host leaves (Figures 53,54). Host plant was classified as Ichnanthus mayarensis Hitchc. Eggs were pale yellow when laid and after 24 hours turned to beige with tiny orange brown spots. 11 additional eggs were obtained on 6–8th April from two females confined to plastic containers. All eggs (n=15) hatched after 6–8 days. Larvae ate the entire corion. First instar (Figure 55)–Head width 0.62–0.68 mm, height 0.58–0.64 mm. Initial length 2.62–3.17 mm, final length 3.70–4.39 mm. Head dark brown, almost black; body pale beige with 7 thin reddish brown longitudinal lines. After feeding on grass background turned pale green. First molt occurred after 9–12 days. Second to fifth instars (Figures 56–61)—After first molt the coloration of larva changed to a new pattern that persisted during subsequent instars with the only variation being a gradual darkening toward final instar. Head width, height, final length and duration of second to fifth instars were: 0.78–0.90, 0.78–0.90, 5.7–6.6 mm, and 7– 10 days at the 2nd (n=15), l.16–1.32, 1.18–1.30, 8.5–9.7 mm, and 6–8 days at the 3rd (n=12), 1.63–1.78, 1.73–1.85, 13.4–14.8 mm, and 7–11 days at the 4th (n=12), and 2.24–2.40, 2.48–2.68, 22–23 mm, and 10–15 days at the 5th, respectively. Head background pale grayish brown heavily speckle in dark brown and with the following dark brown, almost black, marks: lateral vertical lines from stemmata to horns; X shaped mark on epicranium; a complete transverse band across the frontoclypeus. Body background stramineous with an increasing ochre tint toward final instar; lines brown, almost black at final instar; dorsal line with dots on sides at the beginning and end of each segment; subdorsal lines in zig zag with angles closest to dorsal line at the middle of each segment; suprastigmatal line diffuse, present as discontinuous speckling, forming dots over spiracles; spiracle dark brown surrounded by a contrasting white oval shaped area; stigmatal line waved, convex between spiracles; infrastigmatal line straight; sides of last abdominal segment tails white.

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 507 FIGURES 1–8. Types of Calisto from Cuba. Calisto torrei sp. n.: 1—♂ holotype upper side, 2—♂ holotype under side, from Sancti Spiritus, Banao, subida a La Sabina, 450 m, 10/V/2012, DNA voucher RN01–01 (M065), 3—♀ paratype upper side, 4—♀ paratype under side, from Cienfuegos, Pico San Juan, 1140m, 12/V/2012, DNA voucher RN01–03 (M067). Calisto dissimulatum sp. n.: 5—♂ holotype upper side, 6—♂ holotype under side, from Holguín, La Zoilita Station, 400 m, 20/II/2010, DNA voucher PM07–20 (M037), 7—♀ paratype upper side, 8—♀ paratype under side, same data as holotype except 3–10/IV/2012, DNA voucher RN02–01 (M073).

508 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. FIGURES 9–16. Types of Calisto from Cuba. Calisto aquilum sp. n.: 9—♂ holotype upper side, 10—♂ holotype under side, from Sancti Spiritus, Trinidad, ladera norte de Pico Potrerillo, 750–850 m, 11/V/2012, DNA voucher RN01–02 (M070), 11—♀ paratype upper side, 12—♀ paratype under side, same data as holotype. Calisto aquilum occidentalis ssp. n.: 13—♀ holotype upper side, 14—♀ holotype under side, from Pinar del Río, base sur de Pan de Azúcar, 75 m, 25/ IX/2011, DNA voucher PM20–02 (M056), 15—♀ paratype upper side, 16—♀ paratype under side, from Pinar del Río, Sendero Las Maravillas (8 km SW of Viñales village), 200 m, 24/IX/2011, DNA voucher PM20–03 (M057).

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 509 FIGURES 17–22. Live adults of Cuban Calisto. Calisto torrei sp. n.: 17—from Sancti Spiritus, Banao, subida a La Sabina, 450 m, 18—from Sancti Spiritus, Topes de Collante, La Represa, 800 m. Calisto dissimulatum sp. n.: 19, 20— from Holguín, alrededores de La Zoilita, 400m. Calisto aquilum sp. n.: 21—from Cienfuegos, road to Pico San Juan, 22—from Cienfuegos, Pico San Juan.

The prepupal stage lasts three days, two in a straight position, followed by one day in J position until molt. Pupa (Figures 62–67)–Length 10–11 mm, maximum width 4.0–4.5 mm (n=12). Background pale brownish gray with a dark brown spot pattern whose intensity varies between individuals; however constant spots were traced on the available series: a pair of ventral black dots on eyes and on apex of metathoracic tibiae, and first two tarsal segments; a pair of orange dots at the base of labial palpi; a row of tiny dark brown dots on veins near wing outer margin; a waved dark spot at the middle of the wing sheaths, a dark dot near costa at apical third, and a discontinuous series of dark spots on dorsal margin; abdomen with a dark brown line on sides and transverse dark band on dorsum, heavier on the reduced crests of segments 1 to 6; last abdominal segment long, stout, cremaster area enlarged, broad and slightly flattened.

510 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. FIGURES 23–32. Shape and location of androconial patch, and male and female genitalia of Cuban Calisto. Androconial patch: 23—Calisto torrei sp. n., 24—Calisto dissimulatum sp. n., 25—Calisto aquilum sp. n. Male genitalia, from top to bottom main body in lateral view, aedeagus in lateral view, and aedeagus in dorsal view (scale bar 0.5 mm): 26—Calisto torrei sp. n., 27—Calisto dissimulatum sp. n., 28—Calisto aquilum sp. n. Female genitalia, ventral view (scale bar 0.5 mm): 29—Calisto torrei sp. n., 30—Calisto dissimulatum sp. n., 31—Calisto aquilum sp. n. 32—Calisto aquilum occidentalis ssp. n.

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 511 FIGURES 33–36. Geographical distribution of new species of Cuban Calisto. 33—Map of Cuba showing areas where the new taxa were found. 34—Middle rectangle in figure 33, localities where Calisto torrei sp. n. (black circles) and Calisto aquilum sp. n. (white stars) have been found in the Guamuhaya massif. 35—Rigth rectangle in figure 33, localities where Calisto dissimulatum sp. n. has been found in the Sierra Maestra and Nipe–Sagua–Baracoa massifs. 36—Left rectangle in figure 33, localities where Calistoaquilum occidentalis ssp. n. has been found in the middle portion of the Guaniguanico massif.

512 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. The adults emerged between 9:30 am and 4:00 pm from June 6–10th after 9–12 days (n=12). Total developing time was 61–71 days. Remarks. Calisto dissimulatum was another “orphan” lineage found by Núñez et al (2012). It was represented by the specimen coded as PM07-20. At that time the single available specimen led us to confuse it with C. brochei which exhibits a similar wing pattern. More field work showed that this taxon seems to replace C. brochei at the west end of Nipe–Sagua–Baracoa mountain range. The collection of more individuals, including immature stages, and the sequencing of more specimens allow its present description. It was confused with the similar Calisto brochei Torre by Fontenla & Rodríguez (1990) and Núñez et al. (2012). DNA analysis showed large genetic distances of C. dissimulatum from all its Cuban relatives. The closest taxon is the following new species described herein that inhabits the central and western Cuban mountain ranges, with distance values of 4.3 and 4.7% respectively. Another close relative in the Sierra Maestra, C. smintheus (4 specimens, 3 localities, with 0.19% intraspecific variation), is separated by 6.2%. From sympatric C. bruneri (4 specimens, 3 localities, with 0.37% intraspecific variation), C. herophile (10 specimens, 7 localities, 0.8% of intraspecific variation), and C. occulta, C. dissimulatum differs by 8.3, 7.9 and 7.7%, respectively. Intraspecific variation within C. dissimulatum was 0.1% (3 specimens, 1 locality). Recent specimens from localities other than La Zoilita at Sierra Cristal were not available for DNA extraction. Since most known Cuban taxa, excepting C. herophile and the next new species, are restricted to a single mountain range the assignment of other populations to C. dissimulatum must be confirmed, mainly for those inhabiting the western half of Sierra Maestra. The immature stages of C. dissimulatum also showed some diagnostic features. Last larval instar is similar to that C. smintheus but all markings are larger resulting in an overall darker pattern. The majorities of head markings present at C. dissimulatum last instar are reduced or absent in C. smintheus including the vertical lateral lines, the epicraneum X mark, and transverse band of frons. Among all other known taxa, the heavily spotted pattern of pupa is only shared by the closely related C. smintheus.

Calisto aquilum Núñez, sp. n. Figures 9–12, 21–22, 25, 28, 31, 34, 37–40

Type material. Holotype—♂, CUBA, Sancti Spiritus, Trinidad, ladera norte de Pico Potrerillo, 21°53'27"N 80°00'49"W, 750-850 m, 11/V/2012, R. Núñez, DNA voucher RN01–02 (M070). Deposited in CZACC. Paratypes—8 ♂, 2 ♀: same data as holotype, ♂ genitalia in glycerine (3 ♂, 1 ♀). Sancti Spiritus, Loma de Banao, camino de La Sabina a Caja de Agua, 850 m, 11/V/2012, R. Núñez, DNA voucher PM20–01 (M055), genitalia in glycerin (1 ♀). Cienfuegos, Cumanayagua, Pico San Juan, 1140m, 21°59'25"N 80°08'50"W, 12/V/2012, R. Núñez, DNA voucher RN01–07 (M071), ♂ & ♀ genitalia in glycerin (3 ♂, 1 ♀); Cumanayagua, Buenos Aires, 600m, 21°59'13"N 80°11'20"W, 16/VI/1967 (1 ♂); Cumanayagua, carretera a Pico San Juan, V/1986, R. Rodríguez, (1 ♂). Deposited in CZACC. Etymology. The species name is derived from the Latin aquilus (dark–colored, dun), in reference to the dark brownish coloration of the adults. Diagnosis. Calisto aquilum is superficially most similar to C. smintheus, C. brochei and C. dissimulatum¸ a new species previously described herein. From C. smintheus it differs by its less brighter pattern, by lacking the strong reddish suffusion at underside of hindwing, and its smaller average size: ♂ FWL=18.6±0.7 mm, N=7, ♀ FWL=17.9±0.4 mm, N=3 versus ♂ FWL=21.9±1.6 mm, N=50, ♀ FWL=22.5±1.4, N=23. From C. brochei also differs by lacking the waved outer margin of male androconial patch, by its less contrasted underside wing pattern, and its smaller average size: ♂ FWL=20.9±0.8 mm, N=9, ♀ FWL=21.5±0.4, N=5 in C. brochei. From C. dissimulatum differs externally by its less contrasted underside wing pattern. From all three above mentioned species differs by lacking the dense ochre or reddish scaling around subterminal lines at apexes of both wings. Internally, the male genitalia of C. aquilum differ from both C. smintheus and C. brochei by its proportionally smaller uncus when compared to tegumen and it is tapered after basal half instead from base as in C. smintheus and C. brochei. The digitiform projection of valvae is straight as in C. smintheus but shorter whereas it’s slightly curved in C. brochei. There are no appreciable differences with the male genitalia of C. dissimulatum. The female genitalia of Calisto aquilum is diagnostic by having an asymmetric dorsal crown, thinner on the left side in ventral view.

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 513 FIGURES 37–40. Habitats of Calisto torrei sp. n. and Calisto aquilum sp. n. 37—Vicinity of Pico San Juan, 1140 m, at Alturas de Trinidad, the top of the Guamahaya massif, central Cuba. 38—Rainforest understory at Pico San Juan. 39— Broad leaf evergreen forest near La Sabina, Alturas de Banao, Guamuhaya massif. 40–Mogote's (limestome hills) vegetation complex at the slope of Pico Potrerillo (931 m), Alturas de Trinidad, Guamahaya massif.

Description. Forewing length ♂ 17.4–19.8 mm, ♀ 17.3–18.2 mm. Upper surface of wings dark brown, uniform in both sexes; male androconial patch darker than background, almost black, anterior margin behind posterior margin of cell, outer margin parallel to wing margin (Figures 9, 11, 25). Under surface brown with darker lines, background slightly paler at outer margin of post discal line; forewing ocellus with two bluish white iridescent pupils, post discal line externally edged with little contrasting pale yellow scaling at ocellus area; about two fifths of cell extension red colored, both base and apex brown colored as surrounding background; hindwing background mixed with pale yellow and pale ochre scales basal to post dical line which is externally edge by little contrasting pale yellow scaling, heavier around ocellus; post discal area sparsely splashed with lilac scales and four white dots at vein interspaces Rs–M1, M1–M2, M2–M3 and M3–Cu1, dots at M1–M2 and M2–M3 slightly larger than those at Rs–M1 and M3–Cu1 ones (Figures 10, 12, 21, 22). Male genitalia with tegumen about two thirds the length of uncus, slightly concave at the middle and rounded at posterior half; uncus slightly arched, basal half about the same height then gradually tapering toward apex; digitiform projection of valvae, including ventral margin, straight and moderately short; aedeagus sinuated with distinct right curves at both basal and apical halves and an additional smaller curve at the middle (Figure 28). Female genitalia with dorsal crown asymmetric, thinner on the left side in ventral view, height about 0.33x width at right side and 0.20 at the left side; ductus bursae about 1.3x the length of corpus bursae (Figure 31). Distribution. Calisto aquilum is known from localities on both sections, Alturas de Banao and Alturas de Trinidad, of the Guamuhaya massif, the major mountain range of central Cuba (Figures 33, 34). A different subspecies inhabits the Guaniguanico mountain range, in western Cuba. Biology. Collecting sites are at low to moderate elevations (450–1140 m). Habitats include broad leaf evergreen forest, rainforest, and the vegetation complex of the mogotes (limestome hills) (Figures 37–40).

514 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. Individuals have been observed taking nectar on Biden pilosa L. (Asteraceae). Its host plant and immature stages are unknown. Remarks. Genetic differences and geographic isolation from Calisto dissimulatum, were commented at the Remarks section of the latter. From sympatric C. torrei and C. muripetens, C. aquilum differs by 8.5% and 6.5%, respectively. Calisto aquilum is one of the few Cuban taxa present in more than one mountain range. Intraspecific variation of the COI gene suggests that its populations from western and central Cuba differ by 1.4% with an average within populations variation of 0.17 (2 specimens, 2 localities) and 0.11% (3 specimens, 3 localities), respectively. That and a geographic separation of more than 300 km led us to propose the subspecies rank for the western population described below.

FIGURES 41–50. Immature stages of Calisto torrei sp. n. 41—First instar. 42—Second instar. 43—Third instar. 44— Fourth instar. Fifth instar: 45—lateral view, 46—dorsal view, 47—head capsule. Pupa: 48—ventral view, 49—lateral view, 50—dorsal view.

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 515 FIGURES 51–54. Habitats, host plant, and eggs of Calisto dissimulatum sp. n. 51—Pine forest near La Zoilita, Sierra Cristal, Nipe–Sagua–Baracoa massif. 52—Charrascal (scrub forest) near La Zoilita. 53, 54—Ichnanthus mayariensis (Poaceae), host plant with eggs (arrows) at the underside of leaves.

Calisto aquilum occidentalis Núñez, ssp. n. Figures 13–16, 32, 36, 68, 69

Type material. Holotype—♀, CUBA, Pinar del Río, base sur de Pan de Azúcar, 75 m, 25/IX/2011, 22°36'52"N 83°49'27"W, R. Núñez, DNA voucher PM20–02 (M056). Deposited in CZACC. Paratypes—1 ♀: Pinar del Río, Sendero Las Maravillas (8 km SW of Viñales village), 200 m, 24/IX/2011, 22°34'59"N 83°48'15"W, D. Saladrigas & A. Barro, DNA voucher PM20–03 (M057). Deposited in MFP. Etymology. The species name is derived from the Latin occidentalis (of the west), in reference to its distribution restricted to the westernmost Cuban province, Pinar del Río. Diagnosis. Calisto aquilum occidentalis is superficially most similar to Calisto a. aquilum, C. smintheus, C. brochei and C. dissimulatum. From all differs by its smaller average size: ♀ FWL=17.1±0.6, N=2, see size values for other taxa at the Diagnosis of the nominate subspecies. From the nominate subspecies differs by its darker wing pattern and the brighter contrasting underside wing pattern. From all taxa, except Calisto a. aquilum, also differs by having the dorsal crown of the female genitalia asymmetric, thinner on the left side, in ventral view. Description. Forewing length ♀ 16.5–17.6 mm. Upper surface of wings uniform dark brown, almost black (Figures 13, 15). Under surface dark brown with darker lines, background slightly paler at outer side of post discal line; forewing ocellus with two bluish white iridescent pupils, post discal line externally edged with pale yellow scaling at ocellus area; about two fifths of cell extension red colored, both base and apex brown colored as surrounding background; hindwing background mixed with pale yellow and ochre scales basal to post dical line

516 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. which is externally edge by contrasting yellow scaling, heavier around ocellus; post discal area densely splashed with lilac scales and four white dots at vein interspaces Rs–M1, M1–M2, M2–M3 and M3–Cu1, dots at M1–M2 and

M2–M3 slightly larger than those at Rs–M1 and M3–Cu1 ones (Figures 14, 16). Female genitalia with dorsal crown asymmetric, thinner on the left side in ventral view, height about 0.33x width at right side and 0.20 at the left side; ductus bursae about 1.2x the length of corpus bursae (Figure 32). Distribution. Calisto aquilum occidentalis is known only from three localities of the Viñales mountain valley, at the middle portion of the Guaniguanico massif, the major mountain range of western Cuba (Figures 33, 36). Biology. Collecting sites are restricted to the Viñales mountain valley (75–200 m). Species inhabits the semideciduous limestone forest at the base of mogotes (Figures 68, 69). Its host plant and immature stages are unknown. Remarks. The genetic distance between central and western populations (1.4%) of Calisto aquilum is about half the value considered as an indicator of specific differentiation, 2% (Hajibabaei et al. 2006; Huemer & Hebert 2011). Further collecting of more specimens of C. aquilum occidentalis, including males, and the description of their respective immature stages will contribute to confirm these findings. From sympatric C. bradleyi and C. herophile, Calisto aquilum occidentalis differs by 8.6 and 7.1%, respectively.

FIGURES 55–67. Imatures stages of Calisto dissimulatum sp. n. 55—First instar. 56—Second instar. 57—Third instar. 58—Fourth instar. Fifth instar: 59—lateral view, 60—dorsal view, 61—head capsule. Pupa showing variation in spot pattern: 62–64 ventral view, 65, 66—lateral view, 67—dorsal view.

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 517 FIGURES 68–69. Habitats of Calisto aquilum occidentalis ssp. n. 68—Portion of the Sierra de Viñales showing the mogotes covered at the base by semideciduous limestone forest. 69—Understory of the semideciduous limestone forest at “Las Maravillas” path.

FIGURE 70. Phylogenetic hypothesis for Cuban and Bahamian Calisto based on a Bayesian analysis of COI and five nuclear gene regions. Numbers to the right of nodes give the posterior probability of the node. Lineages leading to species are coloured.

518 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. Phylogenetic relationships of Cuban and Bahamian members of Calisto

The Cuban and Bahamian species of Calisto were recovered together as a monophyletic clade with the maximum value of posterior probability (Figure 70). Higher values of posterior probability were also obtained for all the inner nodes with few exceptions. The species group was divided in two major clades. One clade has Calisto israeli Torre 1973 as sister to a clade composed by C. smintheus, C. dissimulatum and C. aquilum. The second and larger clade contains all the remaining Cuban species and the Bahamian ones. Within this clade, the Bahamian Calisto sibylla Bates 1934 and C. bruneri appeared in a separate group, with only 0.56 of posterior probability, as the sister clade of all other species. The following clade groups C. brochei as sister of C. muripetens–C. occulta, with a posterior probability of 0.98. The remaining clade, posterior probability of 1, is divided in two groups. One contains C. h. herophile and at the end of a long branch, derived from the first, the Bahamian C. herophile apollinis. The other has C. torrei as sister of a clade composed by C. bradleyi and a related but apparently different taxon (PM07-06). The latter may represent yet another undescribed species, but since we only have one specimen, we leave that for future investigations.

Discussion

The total number of Calisto species known from Cuba today is 11. This diversity remained hidden by the cryptic nature of most species but also due to the lack of sampling in several regions of the country. Until the 21st century, the number of localities sampled in each major Cuban mountain range was lower than four (Torre 1968, 1973; Fontenla 1987; Fontenla & Rodríguez 1990). In the neighboring Hispaniola, several types of wing patterns can be found among the different species groups whereas in Cuba, with exception of C. israeli, all species exhibit a similar pattern. A population approach, the use of more morphological characters, the discovery and description of their immature stages, and chiefly the use of molecular methods have finally shed light on the taxonomy of the Cuban Calisto. The adults of the newly described species exhibit little morphological variation from their Cuban congeners. Only a few wing and genitalic characters appear to be diagnostic, and that is only after we carefully reviewed a large series of specimens. These features are the same noted by Núñez (2009) and Núñez et al. (2012) for Cuban taxa and for Hispaniolan ones by Johnson et al. (1987), Smith et al. (1994), and Johnson & Hedges (1998). They include the shape of androconial patch, the number and size of white dots on the underside of the hindwing post discal area, the relative size of the tegumen and uncus and the shape of the digitiform projection of valvae in male genitalia, and the height of the dorsal crown and relative length of ductus and corpus bursae in female genitalia. The size of forewing cell area covered by red scaling also proved to be important for distinguishing C. dissimulatum from C. brochei and C. torrei from C. muripetens. Immature stages characters, such as dark marks on the head of last instars and the overall larval and pupal body pattern are also important for taxon recognition. Sourakov (1996, 1999) and Núñez et al. (2012) also used these features among others to recognize each taxon they treated. Defined already by Bates (1935) as the “herophile group”, the Bahamian and Cuban Calisto were recovered in a single monophyletic clade. The group seems to have their origin in northeastern Cuba, with six taxa recorded there presently, expanding later to other Cuban areas and the neighboring Bahamas. Eastern Cuba, together with north central Hispaniola and Puerto Rico has formed a land mass or a chain of close islands since the Paleocene– Eocene (Pindell & Barret 1990; Draper & Barros 1994; Iturralde–Vinent 2006), and that was where the ancestral stock of Calisto possibly began diversifying. One of the two major clades is composed only by Cuban taxa. Calisto israeli is at the base as sister to the remaining members of this lineage. This group evolved into more northeastern taxa, represented today by the sister taxa C. dissimulatum and C. aquilum (which has two subspecies: the western C. a. occidentalis and the central C. a. aquilum). The remaining species, C. smintheus, is found in the geologically younger southern Sierra Maestra. The second and largest clade seems also to have begun evolving in northeastern Cuba. It first split into a small clade of unclear relationships, northeastern Cuban C. bruneri and the Bahamian C. sibylla, and another clade with the remaining taxa. The following clade is divided in two groups. One of these contains the northeastern C. brochei

NEW CALISTO FROM CUBA Zootaxa 3669 (4) © 2013 Magnolia Press · 519 as sister to C. occulta, also northeastern, and C. muripetens, central Cuba. The second one contains a branch with C. torrei from central Cuba at the base and as sister of C. bradleyi, western, and a taxon that seems to represent a potentially new species (specimen PM07-06 in Figure 70). The other branch groups all C. herophile specimens. There is no division justifying the existence of C. herophile parsonsi syn. n. (5 specimens, 2 localities, 0.42% of variation) described from central Guamuhaya massif. The specimens representing these populations (5 specimens, 5 localities, 0.8% of variation) differ from others from localities around the island by a genetic distance of 0.8%. A second colonization event of from Cuba occurred in more recent times apparently from a population of an ancestral taxon related to C. herophile. The present day Bahamian taxon should however be considered as a different species, C. apollinis stat. n., as it is separated from its sister by 2.9%, with 1.4% of intraspecific variation (3 specimens, 2 localities). Further studies on Cuban Calisto must focus on exploring other regions of the island. There are still gaps in our knowledge of the distribution of the genus. These include the eastern extreme of the northeastern mountains which are of a different geological origin, the only true Cuban semi desert along the southeastern coast, and the lower limestone elevations at the north and east of central Cuba among others. Other issues that will require more research involve the search of the immature stages of C. israeli and C. aquilum, an apparent differentiation (1.0%) between the northeastern populations of C. herophile and their conspecifics from other island regions, and also the clarification of the taxonomic status of the southern populations of C. dissimmulatum.

Acknowledgements

We thank Jackie Miller (McGuire Center, Florida) for providing specimens of both Bahamian Calisto for DNA extraction. Niklas Wahlberg acknowledges funding from Kone Foundation. Rayner Núñez acknowledges funding from the Coimbra Group and the Center for International Mobility (TM-12-8194) and to Carlos Peña for his advice in the Laboratory of Genetics of Turku University. We also thank Sara Ilse, David Lambert, Maikel Cañizares, Lucia Echevarría, and Julio for their support during expeditions to Sierra Cristal and the Guamuahaya massif. We also thank Luis Catasús and Ramona Oviedo for their help in host plant and nectar source identification, respectively. We thank also Andrei Sourakov and a second anonymous referee for reviewing the article and making helpful corrections and comments.

Literature cited

Bates, M. (1934) New Lepidoptera from the Bahamas. Occasional Papers Boston Society of Natural History, 8, 133–138. Bates, M. (1935) The satyrid genus Calisto. Occasional Papers Boston Society of Natural History, 8, 229–248. Brown, F.M. & Heineman, B. (1972) Jamaica and its butterflies. London, E.W. Classey Ltd., xv + 478 pp. Dethier, V.G. (1940) Life histories of Cuban Lepidoptera. Psyche, 47, 14–26. http://dx.doi.org/10.1155/1940/78098 Draper, G. & Barros, J.A. (1994) Cuba. In: Donovan, S.K. & Jackson, T.A. (Eds.), Geology: an Introduction. University of the West Indies, Kingston, pp. 65–86. Fontenla, J.L. (1987) Características zoogeográficas de las ropalóceras (Insecta: Lepidoptera) de Viñales, Pinar del Río, Cuba. Poeyana, 339, 1–11. Fontenla, J.L. & Rodríguez, R. (1990) Sistema de poblaciones de Calisto sibylla Bates, 1934 (Lepidoptera, Satyridae) en Cuba. Poeyana, 395, 1–13. Hajibabaei, M., Janzen, D.H., Burns, J.M., Hallwachs, W. & Hebert, P.D.N. (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proceedings of the National Academy of Sciences, 103 (4), 968–971. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98. Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270 (1512), 313–321. http://dx.doi.org/10.1098/rspb.2002.2218 Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H. & Hallwachs, W. (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences, 101, 14812–14817. http://dx.doi.org/10.1073/pnas.0406166101 Hernandez, L.R. (2004) Field Guide of Cuban–West Indies Butterflies. EDILUZ, Maracaibo, Venezuela. 269 pp., 31 pl.

520 · Zootaxa 3669 (4) © 2013 Magnolia Press NÚÑEZ AGUILA ET AL. Huemer, P. &. Hebert, P.D.N. (2011) Cryptic diversity and phylogeography of high alpine Sattleria–a case study combining DNA barcodes and morphology (Lepidoptera: Gelechiidae). Zootaxa, 2981, 1–22. Iturralde–Vinent, M.A. (2006) Meso-Cenozoic Caribbean Paleogeography: Implications for the Historical Biogeography of the Region. International Geology Review, 48, 791–827. http://dx.doi.org/10.2747/0020-6814.48.9.791 Johnson, K. & Hedges, S.B. (1998) Three new species of Calisto from Southwestern Haiti (Lepidoptera: Nymphalidae: Satyrinae), Tropical Lepidoptera, 9 (2), 45–53. Johnson, K., Quinter, E.L. & Matusik, D. (1987) A new species of Calisto from Hispaniola with a review of the female genitalia of Hispaniola congeners (Satyridae). Journal of Research on the Lepidoptera, 25 (2), 73–82. Lamas, G. (Ed.) (2004) Checklist: Part 4A. Hesperioidea–Papilionoidea. Atlas of Neotropical Lepidoptera. Association for Tropical Lepidoptera/Scientific Publishers, 439 pp. Michener, C.D. (1949) Sympatric species of Calisto in Cuba (Lepidoptera, Satyrinae). American Museum Novitates, 1391, 1–3. Miller, J.Y. & Miller L.D. (2001) 10. The Biogeography of the West Indian Butterflies An Application of a Vicariance/ Dispersalist Model. In: Woods, C.A. & Sergile, E. (Eds.), Biogeography of the West Indies: patterns and perspectives. 2nd edition. CRC Press, Boca Raton, pp. 127–155. Núñez, R. (2009) Rediscovery of Calisto israeli Torre, with nomenclatural notes on the larger species of Cuban Calisto (Lepidoptera: Nymphalidae: Satyrinae). Zootaxa, 2087, 46–58. Núñez, R., Oliva, E., Matos, P.F. & Wahlberg, N. (2012) Cuban Calisto (Lepidoptera, Nymphalidae, Satyrinae), a review based on morphological and DNA data. ZooKeys, 165, 57–105. http://dx.doi.org/10.3897/zookeys.165.2206 Pindell, J. & Barrett, S.F. (1990) Geological evolution of the Caribbean: a plate tectonic perspective. In: Dengo, G. & Case, J.E. (Eds.), The Geology of North America, Vol. H, The Caribbean Region. Geological Society of America, Boulder, pp. 405– 432. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539–542. http://dx.doi.org/10.1093/sysbio/sys029 Smith, D.S., Miller, L.D. & Miller, J.Y. (1994) The Butterflies of the West Indies and South Florida. Oxford University Press, New York, 284 pp. Sourakov, A. (1996) Notes on the genus Calisto, part 1. Tropical Lepidoptera, 7 (1) part 2, 91–111. Sourakov, A. (1999) Notes on the genus Calisto, with descriptions of the immature stages (part 2) (Lepidoptera: Nymphalidae: Satyrinae). Tropical Lepidoptera 10 (2), 73–79. Sourakov, A. & Emmel, T.C. (1995) Early stages of Calisto grannus Bates on Hispaniola (Lepidoptera: Nymphalidae: Satyrinae). Tropical Lepidoptera, 6 (1), 27–30. Sourakov, A. & Zakharov, E.V. (2011) “Darwin’s butterflies?” DNA barcoding and the radiation of the endemic Caribbean butterfly genus Calisto (Lepidoptera, Nymphalidae, Satyrinae). Comparative Cytogenetics, 5 (3), 191–210. http://dx.doi.org/10.3897/compcytogen.v5i3.1730 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731–2739. http://dx.doi.org/10.1093/molbev/msr121 Torre, S.L. de la. (1968) Revisión de las especies cubanas de la familia Satyridae (Lepidoptera, Rhopalocera), con la descripción de una nueva especie. Ciencias Biológicas serie 4, 3, 1–24. Torre, S.L. de la. (1973) Una nueva especie del género Calisto Hübner y una nueva raza. Ciencias Biológicas serie 4, 43, 1–9. Wahlberg, N. & Wheat, C.W. (2008) Genomic outposts serve the phylogenomic pioneers: designing novel nuclear markers for genomic DNA extractions of lepidoptera. Systematic Biology, 57 (2), 231–242. http://dx.doi.org/10.1080/10635150802033006

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