The Heliconiini genera Agraulis and Dryas are widely distributed throughout the Neotropics and into adjacent temperate regions, and although they are currently treated as monotypic, both show significant geographic phenotypic variation. In this work, we employ six genetic markers (4199 bp), two mitochondrial and four nuclear, to perform coalescent species delimitation analyses in Bayesian Phylogenetics and Phylogeography (BPP) and in integrated BPP (iBPP), the latter also includes linear measurements and wings landmarks. We also analyze cytochrome c oxidase I (COI) barcode sequences for each genus using genetic distances, haplotype networks and a character-based approach. Based on the model testing results, complemented with data from previous studies, we performed morphometric analyses to compare fore and hindwing size, aspect ratio and shape among the new species. In addition, we compared the forewing spot pattern of hypothesizes species using the r package patternize and, for Dryas, compared the colour patterns of mature larvae. Model testing of the molecular species delimitation outputs favoured a seven species hypothesis for Agraulis and a four species hypothesis for Dryas. Average distances among COI barcode sequences of these groups were from 1.09 to 5.81% in Agraulis and from 1.09 to 3.44% in Dryas. Within-group distances ranged from 0 to 1.11% and between 0 and 2.43%, respectively. NeighborNet haplotype networks showed that all but one of the species are monophyletic, and the character-based approach found exclusive diagnostic positions for most species, while the rest can be recognized by unique combinations of the 44 informative nucleotide positions analysed. Morphometric analysis supported all species of Agraulis and Dryas based on wing shape, and also in several cases on wing sizes and aspect ratio (hindwing length-forewing length), including A. v. galapagensis, which was absent from the molecular study. The analysis of the forewing spot pattern also revealed differences among most species hypothesis.

The most accepted taxonomic treatment of the New World sulphurs of the genus Phoebis Hübner, [1819] recognizes 16 species including those in the current synonyms Aphrissa and Rhabdodryas. This total conflicts with the results of several recent pierid DNA barcode studies across the Neotropics. We used a five‐locus dataset to carry out species delimitation analyses using the coalescence‐based method implemented in bpp software. After testing the resulting species hypotheses using marginal likelihood estimates, we inferred their phylogenetic relationships and performed an ancestral range reconstruction with biogeobears. Our analyses recovered two different hypotheses, 26 and 24 species, that scored the highest marginal likelihood estimate. Differences between these two hypotheses, when reconciled with barcode clusters and morphology, indicated that 24 is the most likely number of species. Phoebis neocypris stat. rev., Phoebis rurina stat. rev., Phoebis virgo stat. rev., Phoebis marcellina stat. rev., Phoebis thalestris stat. rev., and Phoebis rorata stat. rev. are raised to the species rank. We dated the crown age of Phoebis to the mid‐Miocene, with the islands of the Greater Antilles as the most probable ancestral range. Three main clades of Phoebis diverged early in the evolutionary history of the genus, but most extant species‐level diversity arose after the Pliocene–Pleistocene boundary. Our analyses recovered alternate range expansions and contractions, and dispersal from the islands to the continent and back, in the three main clades. Both sympatric and allopatric speciation seem to have shaped the current species richness.

In this work, we used mtDNA data as a tool to delimit species and we compared the resulting molecular operational taxonomic units (barcode index number, BIN) with morphology-based identifications in the Colombian species of Rhamma Johnson, 1992 exploring the usefulness of DNA barcodes for taxonomy, species identification and delimitation. We obtained cytochrome oxidase I (COI) sequences for 134 morphologically identified specimens, representing 12 species of Rhamma from Colombia. Ten of these species have not been previously barcoded. DNA barcodes suggested the potential for eight additional cryptic species in Colombia but we were readily able to morphologically diagnose just one of these linages as a new species which recently was described in a separate paper as Rhamma dawkinsi Prieto & Lorenc-Brudecka, 2017. The morphological species were separated into three categories: species showing a perfect match between morphological species and BINs (33%, four species); species sharing a BIN completely or partly (single specimens) with another morphological species (42%, five species placed in three BINs); and morphological species splitting up into more than one BIN (25%, three species placed in 10 BINs). The high percentages of incongruence between morphology-based identification and species delineation through BINs, could be explained as a consequence of high rates of introgressive hybridization. However, DNA barcodes can be considered diagnostic even in cases where specimens of a species were assigned to two or more distinct BINs and in species showing a low but constant divergence causing their assignment to a single BIN, which is often the case in young, allopatric species. We retain 10 of the 12 species (83%) to be diagnostic in molecular identification.

The genus Calisto is endemic tothe West Indiesand the only representative there of the Satyrinae. Here wereconstruct the evolutionary relationshipsof the herophile group and describe five new species from Cuba: Calisto gundlachi sp. nov., Calisto siguanensis sp. nov., Calisto disjunctus sp. nov., Calisto sharkeyae sp. nov. and Calisto lastrai sp. nov.We employ one mitochondrial and four nuclear markers to assess the phylogenetic position, Maximum Likelihood and Bayesian Inference approaches, of the new taxa. Our phylogenetic trees yielded two strongly supported main clades with four of the new species included within them and C. sharkeyae as sister group to the rest of the major main clade. We conduct time-divergence estimations and ancestral area reconstructions using BEAST and BioGeoBEARS. The group originated 12.15 million years ago during the middle Miocene in north-eastern Cuba, Nipe-Sagua-Baracoa Massif. After 6 million years of in situ evolution most lineages started to colonise other Cuban territories and the Bahamas. This scenario is consistent with key geological events, including the closure of the western Havana–Matanzas channel 8–6 million years ago, the uplift of the Sierra Maestra 6–5 million years ago, and the land connections among Cuban regions during the Miocene–Pleistocene sea level drops. Dispersal and vicariance processes may have occurred, with populations surviving floodings on the major and minor mountain ranges, which remained as ‘islands’. http://zoobank.org/urn:lsid:zoobank.org:act:03690F79-F938-42A0-B234-4A228D5C1913

In the present work we reassess the taxonomic status of one species of Hesperiidae, subfamily Pyrginae, originally described from Cuba. As part of a larger project on the Greater Antillean butterflies, we obtained COI barcode sequences for Cuban specimens of Chiomara mithrax (Möschler, 1879). The comparison of these sequences revealed deep divergences from others belonging to continental specimens. Inspired by these results we performed morphological comparisons including genitalia structures, which revealed correlating differences between specimens from Cuba and the continent. Therefore, we propose the necessary changes in its taxonomy.DNA extraction, PCR amplification, and sequencing of the COI barcode region were performed by the Canadian Centre for DNA Barcoding (CCDB). Polymerase chain reactions used the primer pair LepF1-ATTCAACCAATCATAAAGATATTGG and LepR1-TAAACTTCTGGATGTCCAAAAAATCA (Hebert et al. 2004) which recovers a 658 bp region near the 5´ end of COI including the 648 bp barcode region for the animal kingdom (Hebert et al. 2003). Our two new sequences were submitted to GenBank: MK253654-55. COI barcode sequences of continental C. mithrax were downloaded from GenBank: JF777766, GU658613-14, GU658617-20, GU658246.. We examined adult specimens deposited at the Zoologische Staatssammlung München (ZSM), Munich, Germany and the Institute of Ecology and Systematics (CZACC) in Havana, Cuba. Genitalia dissections were made using standard techniques. Abdomens were soaked in hot 10% potassium hydroxide for one hour before dissection. The structures were then dehydrated in an alcoholic series and mounted on slides using Euparal. FIGURES 1-3. Adults of Chiomara gundlachi stat. rev. 1-Outskirts of Vallecito village, Pinar del Río, Cuba. 2-same data, road kill. 3-La Movida, outskirts of Santa Clara city, Villa Clara Cuba. Photos: Tim Norriss.FIGURES 4-6. Adults of Chiomara mithrax. 4-Chiriqui, Panama. 5-Sinaloa, Mexico. 6-Rio Grande do Sul, Brazil. Scale bar 10 mm.

Calisto disjunctus hersheyi ssp. n. is described from western Cuba. Additionally, a dichotomous key including all described Cuban taxa of the herophile species group of Calisto is given together with illustrations of live specimens of most taxa.

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