Getting western: biogeographical analysis of morphological variation,mitochondrial haplotypes and nuclear markers reveals cryptic species and hybrid zones in the Junonia butterflies of the American southwest and Mexico

The American southwest and northern Mexico has a great degree of endemic diversity compared with the rest of North America. The Pleistocene glaciations and the dispersal of species from glacial refuges in this region have been important engines for the production of biodiversity in the region. The New World Junonia are a recent radiation of butterflies that are thought to have spent time in these refuges during periods of glacial advancement. We have reconstructed the plausible movements and the contemporary geographic distributions of the five taxa (J. coenia, J. grisea, J. litoralis, J. nigrosuffusa and J. zonalis) that occur in the American southwest and northern Mexico using phenotypic and genotypic information primarily from specimens preserved in museum collections, supplemented with additional contemporary collections. Evidence of cryptic species and hybridization events were observed using mitochondrial haplotypes, genotypic variation at the nuclear wingless locus, multilocus DNA fingerprinting, patterns of larval host plant use, variation in life‐history traits and morphological characteristics. Based on these lines of evidence, and in spite of low levels of hybridization between them, we argue that all five Junonia taxa are independent evolutionary lineages. Junonia grisea and J. coenia are morphologically very similar, but differences in morphology, life‐history traits, nuclear genotypes and mitochondrial haplotypes suggest that they are a cryptic species pair, thus elevating J. grisea comb.n. to a full species when it had previously been considered to be a subspecies of J. coenia.     

Taxonomy as a hypothesis: testing the status of the Bermuda buckeye butterfly Junonia coenia bergi (Lepidoptera: Nymphalidae)

Species determination and definition in eukaryotes have traditionally been based on morphology, with little focus on genetic differentiation. Molecular methods allow for the independent assessment of morphology‐based taxonomic hypotheses. Three criteria used to define a full species for taxonomic purposes are morphological distinction, formation of a monophyletic lineage, and reproductive isolation. Junonia butterflies (Nymphalidae) are becoming an important experimental model system, but the taxonomy of many New World Junonia species is unclear. One of these species is J. coenia, which contains the subspecies J. coenia coenia, J. coenia grisea and J. coenia bergi. Previous studies suggest that J. coenia grisea may meet the criteria for full species status. Therefore, we evaluated the geographically isolated and rarely studied Bermuda buckeye butterfly J. coenia bergi to determine if it was similarly distinct. Physical examination of specimens and phylogenetic and population genetic analyses of mitochondrial cytochrome c oxidase subunit I, nuclear wingless, and complete mitochondrial genome sequences suggest that while J. coenia bergi is smaller in body size than many Junonia and has distinctive ventral hindwing colouration, it does not form a monophyletic lineage and shows indications of continued gene flow with North American mainland J. coenia coenia populations. Thus, J. coenia bergi does not meet the criteria for full species designation, but geographic isolation, morphological distinctiveness, and cultural importance suggest that it remain recognized as a subspecies of J. coenia. Similar analyses will be useful for addressing further taxonomic questions in Junonia and other taxa, especially where morphology‐based taxonomic determinations are ambiguous.     

Genetic population structure of buckeye butterflies (Junonia) from Argentina

There are nine named species of buckeye butterflies (genus Junonia Hübner) in the Western Hemisphere. There is considerable geographic variation within Junonia species, and possible ongoing hybridization between species, suggesting that Junonia may be a ring species, but also making this a very difficult group to define taxonomically. We tried to determine whether two forms of Junonia from Argentina – conventionally referred to as Junonia genoveva hilaris C. & R. Felder, the light buckeye butterfly, and Junonia evarete flirtea (Fabricius), the dark buckeye butterfly – were genetically distinct species or simply colour forms of a single species using morphological characters, mitochondrial cytochrome oxidase I (COI) DNA barcodes, nuclear wingless (wg) locus DNA sequences, and anonymous nuclear Randomly Amplified Fingerprints (RAF). Phylogenetic analysis of COI identified two distinct mitochondrial haplotypes that differ by about 4% sequence divergence; one confined to light‐coloured Junonia specimens and one shared between some light‐coloured Junonia and all of dark‐coloured Junonia specimens. Analysis of nuclear wingless sequences revealed 32 alleles among 22 Junonia specimens and showed significant genetic differentiation between light‐coloured and dark‐coloured Junonia. Analysis of RAF genotypes suggests that there are actually three genetically distinct Junonia populations in Argentina: two with light wing coloration, and one with dark wing coloration. Genetic evidence of recent hybridization among these populations was also observed, consistent with the ring species hypothesis. Careful comparisons of morphological characters between Argentinian Junonia and Junonia species from elsewhere in South America suggests that the two light‐coloured populations correspond to J. genoveva and either a genetically disparate population of the same species or an undescribed cryptic Junonia species, The dark‐coloured population may correspond to J. wahlbergi Brévignon. Our data suggest that COI DNA barcodes by themselves are not very useful for studying Junonia taxonomy, population structure or evolution.     

Relative susceptibility to predation of two species of caterpillar on plantain

Summary The relative susceptibility to predators of a cryptic generalist caterpillar (Spilosoma congrua: Arctiidae) and a non-cryptic specialist (Junonia coenia: Nymphalidae) using the same hostplant species (Plantago lanceolata) was examined. In a laboratory experiment using predatory stinkbugs (Podisus maculiventris), more Junonia caterpillars than Spilosoma caterpillars were killed (70% vs. 16%). This result was a consequence of the Spilosoma spending some time under cover, moving frequently, feeding on leaves while under or adjacent to them, and spending little time on the leaves. In a field experiment using predatory wasps (Polistes fuscatus), the wasps found 7 times as many of the Junonia as the Spilosoma, and overall 6 times as many Junonia were killed as Spilosoma. Initially, 71% of the Junonia caterpillars encountered by wasps were killed, but by the fourth day of the test, only 22% of the Junonia encountered by wasps were killed. Over three full days of observations, a constant 50% of the Spilosoma caterpillars encountered by the wasps per day were killed. For the Junonia, evasion of predators rested on passive chemical defense. For the Spilosoma, evasion depended on being unapparent, speedy movement between feeding and resting sites and, if found, on fleeing immediately and quickly. These results indicate that Spilosoma caterpillars, by way of cryptic and escape behaviors, can be less susceptible to insect predators than Junonia caterpillars.     

Dietary specialization and the effects of plant species on potential multitrophic interactions of three species of nymphaline caterpillars

The diet breadth of insect herbivores influences their response to variation in plant quality, and these bitrophic interactions have implications for the higher‐level trophic interactions between herbivores and their natural enemies. In this comparative study, we examined the role of host plant species and plant secondary chemistry on the potential interactions between three species of nymphaline caterpillars and their natural enemies. The caterpillar species (all Lepidoptera: Nymphalidae) varied in their degree of specialization: the buckeye, Junonia coenia Hübner, is a specialist on plants that contain iridoid glycosides (IGs); the white peacock, Anartia jatrophae L., feeds on plants in five families, some of which contain IGs and some of which do not; and the painted lady, Vanessa cardui L., is a generalist, feeding on plants in at least 15 families. Each species was reared on leaves of an introduced host plant, Plantago lanceolata L. (Plantaginaceae), which produces two IGs, aucubin and catalpol, and on another plant species that is a common host plant. These alternate host plants were Plantago major L. (Plantaginaceae) for J. coenia, Bacopa monnieri (L.) Pennell (Plantaginaceae) for A. jatrophae, and Malva parviflora L. (Malvaceae) for V. cardui. We examined growth, sequestration, and immune response of these caterpillars on the different host plant species. Junonia coenia developed more rapidly and sequestered higher IG concentrations when reared on P. lanceolata, whereas both other species grew more slowly on P. lanceolata. Host plant did not influence immune response of J. coenia or A. jatrophae, whereas V. cardui immune response was weaker when reared on P. lanceolata. Junonia coenia was most efficient at IG sequestration and A. jatrophae was least efficient, when all three species were reared on P. lanceolata. These results indicate that diet breadth may play an important role in structuring tritrophic interactions, and this role should be further explored.     

Effects of prey quality on social wasps when given a choice of prey

The effect of prey quality on foraging behavior and colony demographics of the social wasp Polistes fuscatus was examined by providing a choice between non‐toxic prey (Manduca sexta caterpillars) and sublethally toxic prey (Junonia coenia caterpillars), and then comparing the performance of these colonies to others given only the non‐toxic prey. In the choice, one of two types of Manduca were used: those fed an artificial diet or those fed plantain (Plantago lanceolata), which contains iridoid glycosides (IGs) that Junonia coenia store but which Manduca does not. Despite the negative correlation between the number of Junonia prey used and number of adult offspring produced, when a surplus of non‐toxic prey was available, the wasps did not completely avoid the toxic prey. However, they were more discriminating when the choice was between Manduca fed an artificial diet and Junonia fed plantain vs. when both prey species had eaten the plantain. Because the wasps had a choice of prey types and had a surplus of prey on about one‐third of the days, the wasps were able to take enough non‐toxic prey to avoid some of the negative consequences of IGs. For example, the total number of wasp offspring per nest was not affected, but mean weight of female offspring per colony was less for colonies given both prey types eating plantain, compared to that for colonies fed only non‐toxic prey, or those given a choice of non‐toxic prey vs. toxic prey. In addition, compared to the control (only non‐toxic prey), the proportion of males produced was less in the treatment that provided a clear contrast between non‐toxic and toxic prey. Why these wasps did not avoid the toxic prey is discussed.     

Larval feeding experience influences adult predator acceptance of chemically defended prey

Generalist predatory paper wasps, Polistes dominulus, experience plant secondary defensive compounds as developing larvae through their herbivorous lepidopteran caterpillar prey and as adults through attacking caterpillars while foraging. We evaluated the role that larval and early adult experience with unpalatable prey plays in subsequent foraging choices by adult wasps. For periods of two or four weeks, caged wasps were raised exclusively on caterpillars of either unpalatable Buckeye, Junonia coenia, that sequester substantial levels of iridoid glycosides (IG) or on Painted Lady, Vanessa cardui, that contain very low levels of IG. Wasps were then allowed to forage on both caterpillar species simultaneously. Patterns of prey capture differed significantly based on previous prey experience. Regardless of previous feeding experience, adult wasps overwhelmingly preferred to take Vanessa. Yet Junonia-experienced wasps continued to attack and take back to the nest over 50% more Junonia than did Vanessa-experienced wasps. The longer the wasps' larval experience with Junonia, the more likely they were to capture Junonia caterpillars. However, the life stage at which the wasps experienced Junonia was also influential as young adult experience with the unpalatable prey was more of a deterrent than was experience strictly as larvae for Junonia-experienced wasps. The results demonstrate that, in these predators, previous experience with deterrent chemicals during their larval development alters patterns of prey acceptability to the adult insects.     

Presence of predatory wasps and stinkbugs alters foraging behavior of cryptic and non-cryptic caterpillars on plantain (Plantago lanceolata)

We examined the foraging patterns of two species of caterpillar (Junonia coenia: Nymphalidae and Spilosoma congrua: Arctiidae) that contrast in feeding specialization and crypticity on plantain (Plantago lanceolata) in the absence and presence of two different insect predators [stinkbugs, Podisus maculiventris (Pentatomidae) and wasps, Polistes fuscatus (Vespidae)]. Junonia larvae were quite apparent to human observers, feeding on upper leaf surfaces during daylight, whereas Spilosoma larvae were relatively cryptic, often hiding under leaves and in soil crevices during daylight. In the presence of either predator species, the non-cryptic Junonia caterpillars more quickly left the plant on which they were initially placed and were less apparent than Junonia larvae not exposed to predators. The presence of predators had no detectable influence on where the caterpillars occurred on the plants (new, intermediate-aged or mature leaves, or reproductive stalks). Surprisingly, the predators influenced the behavior of the inherently cryptic Spilosoma: the apparency of these larvae at night increased when wasps had access to the plots during the day. Survivorship of the non-cryptic Junonia was less than 12% when stinkbugs were present compared to 60% in their absence. Although the presence of wasps resulted in a lower relative growth rate for the non-cryptic Junonia larvae, the indirect effect of predators on reduction in survivorship due to alterations in prey growth rate through behavioral changes was less than 3%. After taking into account the decline in caterpillars per plot through predation, we found that both the amount of leaves eaten and the proportion of plants eaten were altered on plots with predators present, which suggests that the caterpillars' increased consumption countered increased maintenance costs due to the presence of predators. Overall, our results indicate that hostplant size, level of predation and type of predator can influence the degree to which these caterpillars react to the presence of insect predators. In contrast, degree of inherent feeding specialization and cryptic behavior seemed to have little effect on the expression of reactive behaviors of these caterpillars to predators.     

Phylogenetic relationships and historical biogeography of tribes and genera in the subfamily Nymphalinae (Lepidoptera: Nymphalidae)

We infer for the first time the phylogenetic relationships of genera and tribes in the ecologically and evolutionarily well‐studied subfamily Nymphalinae using DNA sequence data from three genes: 1450 bp of cytochrome oxidase subunit I (COI) (in the mitochondrial genome), 1077 bp of elongation factor 1‐alpha (EF1‐α) and 400–403 bp of wingless (both in the nuclear genome). We explore the influence of each gene region on the support given to each node of the most parsimonious tree derived from a combined analysis of all three genes using Partitioned Bremer Support. We also explore the influence of assuming equal weights for all characters in the combined analysis by investigating the stability of clades to different transition/transversion weighting schemes. We find many strongly supported and stable clades in the Nymphalinae. We are also able to identify ‘rogue’ taxa whose positions are weakly supported (the different gene regions are in conflict with each other) and unstable. Our main conclusions are: (1) the tribe Coeini as currently constituted is untenable, and Smyrna, Colobura and Tigridia are part of Nymphalini; (2) ‘Kallimini’ is paraphyletic with regard to Melitaeini and should be split into three tribes: Kallimini s.s., Junoniini and Victorinini; (3) Junoniini, Victorinini, Melitaeini and the newly circumscribed Nymphalini are strongly supported monophyletic groups, and (4) Precis and Junonia are not synonymous or even sister groups. The species Junonia coenia, a model system in developmental biology, clearly belongs in the genus Junonia. A dispersal‐vicariance analysis suggests that dispersal has had a major effect on the distributions of extant species, and three biotic regions are identified as being centres of diversification of three major clades: the Palaearctic for the Nymphalis‐group, the Afrotropics for Junoniini and the Nearctic for Melitaeini. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86 , 227–251.     

Two new species of Apsilochorema Ulmer, 1907 (Trichoptera: Hydrobiosidae) from India

Two new species Apsilochorema (Apsilochorema) shalimarensis sp. n. and Apsilochorema (Apsilochorema) sainii sp. n. are described and illustrated from India. With these new additions, the genus Apsilochorema Ulmer, 1907 is now represented by 14 species from India.http://www.zoobank.org/urn:lsid:zoobank.org:pub:AAA8C469-850E-4778-BCF1-3FF5B429E559     

New species of the subfamily Conoderinae (Coleoptera: Curculionidae) in Dominican amber

Eulechriops argyrosoman. sp. (Curculionidae: Conoderinae: Lechropini) and Geratozygops platysoman. sp., Geratozygops stenosoman. sp. and Geratozygops arsinotusn. sp. (Curculionidae: Conoderinae: Zygopini) are described from Dominican amber. The small size, nearly parallel sides of the pronotum and silvery sheen distinguish Eulechriops argyrosoma from extant members of the genus, which have not been recorded from Hispaniola. Size and rostral and pronotal characters separate the three Geratozygops species from the single species (Geratozygops atropos) previously described from Dominican amber.http://www.zoobank.org/urn:lsid:zoobank.org:pub:89BC7F43-6C1A-4A6A-839B-CE94C7F6F140     

An integrative approach elucidates the systematics of Sea Hayward and Cybdelis Boisduval (Lepidoptera: Nymphalidae: Biblidinae)

The systematics of the enigmatic and rather uncommon species of Sea Hayward and Cybdelis Boisduval is assessed through morphological and molecular analyses. The aim of this paper is three‐fold: (i) to ascertain the phylogenetic position of Sea and Cybdelis in Biblidinae; (ii) to determine the evolutionary relationships among species of Sea and Cybdelis; and (iii) to review the taxonomy of Sea and Cybdelis. The result of the molecular analysis recovers Sea sister to Cybdelis, and this clade sister to Eubagina (sensu Lamas, 2004 ), rendering Epicaliina (sensu Lamas, 2004 ) paraphyletic. A new tribe, Cybdelini trib.n. is erected to include Sea and Cybdelis; the other genera formerly included in Epicaliina (sensu Lamas, 2004 ), Eunica Hübner and Sevenia Koçak are assigned to Eunicini trib.rest. , and the remaining genera of Biblidinae to other previously recognized tribes, similar to those implicitly proposed by Wahlberg et al. ( 2009 ). The morphological analysis recovers similar results and the following relationships among species of Cybdelis: C. phaesyla (Hübner) (C. boliviana Salvin (C. mnasylus Doubleday (C. petronita Burmeister stat.rest. + C. thrasylla C. Felder & R. Felder stat.rest. ))). Sea and Cybdelis are recognized as distinct genera; a new subspecies, S. sophronia jenkinsi Dias, Siewert & Freitas ssp.n. , distributed from Venezuela to Bolivia along the Andes eastern foothills, is described; and C. petronita and C. thrasylla are accepted as valid species. Lectotypes for C. boliviana, C. peruviana Staudinger, C. mnasylus, C. thrasylla, C. petronita and C. mnasylus var. meridionalis Hall and a neotype for Vanessa sophronia Godart are designated. Additionally, dichotomous keys and distribution maps to all taxa are provided; male genitalia and hypandria are illustrated for all species; generic characters of the head, labial palpi and legs are illustrated; immature stages of some species also are figured. The published work has been registered on ZooBank: http://zoobank.org/urn:lsid:zoobank.org:pub:D8EBB649‐5E9D‐4218‐B1F1‐7A69DA775FD0 .     

Description of two new species of Pseudamnicola from southern Turkey (Mollusca: Gastropoda: Hydrobiidae)

New material of freshwater molluscs in the Mediterranean region of Turkey revealed two new species of the genus Pseudamnicola Paulucci, 1878, i.e., P. kayseriensis n. sp., and P. gullei n. sp.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:3E39E467-22A5-4091-9975-17343DA8D02E     

A tale of two haplotype groups: evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes

The New World Junonia butterflies are a possible ring species with a circum‐Caribbean distribution. Previous reports suggest a steady transition between North and South American forms in Mesoamerica, but in Cuba the forms were thought to co‐exist without interbreeding representing the overlapping ends of the ring. Three criteria establish the existence of a ring species: a ring‐shaped geographic distribution, gene flow among intervening forms and genetic isolation in the region of range overlap. We evaluated mitochondrial cytochrome oxidase I haplotypes in Junonia from nine species in the Western Hemisphere to test the Junonia ring species hypothesis. Junonia species are generally not monophyletic with respect to COI haplotypes, which are shared across species. However, two major COI haplotype groups exist. Group A predominates in South America, and Group B predominates in North and Central America. Therefore, COI haplotypes can be used to assess the degree of genetic influence a population receives from each continent. Junonia shows a ring‐shaped distribution around the Caribbean, and evidence is consistent with gene flow among forms of Junonia, including those from Mesoamerica. However, we detected no discontinuity in gene flow in Cuba or elsewhere in the Caribbean consistent with genetic isolation in the region of overlap. Although sampling is still very limited in the critical region, the only remaining possibility for a circum‐Caribbean discontinuity in gene flow is at the Isthmus of Panama, where there may be a transition from 98% Group B haplotypes in Costa Rica to 85–100% Group A haplotypes in South America.     

On the genus Sunius Curtis, 1829 of Turkey III. Four new species from western Anatolia,Turkey (Coleoptera: Staphylinidae: Paederinae)

Four new micropterous species of the genus Sunius Curtis, 1829 are described and illustrated from western Anatolia: Sunius akdaghensis sp. n. from Kütahya province, Sunius ciceki sp. n. from Bal?kesir province, Sunius ozgeni sp. n. and Sunius cagatayi sp. n. from Denizli province. Additional records are presented for three species. The genus Sunius is now represented in Turkey by 36 species.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:F3808D28-65E9-4619-A350-781C971701EC     

Prozercon iranensis sp. n., a new species of Zerconidae (Acari: Mesostigmata) from Iran

A new species of pseudoscorpion of the genus Calocheiridius Beier &; Turk, Calocheiridius iranicus n. sp., is described from Markazi province, Iran. Illustrations and measurements of the species are given. http://www.zoobank.org/urn:lsid:zoobank.org:pub:06332B5C-D43A-4955-AB6C- 5847D10C2BAC     

A new species of weevil of the genus Rhytideres (Coleoptera: Entiminae: Alophini) from Inner Anatolia (Turkey)

A new species, Rhytideres evrani Erbey sp. n., is described from Inner Anatolia (Turkey), which is closely related to R. plicatus Olivier, 1790.

http://www.zoobank.org/urn:lsid:zoobank.org:pub: AF936EEE-D6F2-4830-9D42-35CEF250E066     

Hydraenidae (Insecta: Coleoptera) of Mindoro,Philippines. I: Hydraena Kugelann, 1794 of the Baroc River basin,Roxas, Oriental Mindoro with description of three new species

The species of the genus Hydraena Kugelann, 1794 of the Baroc River basin in the Philippine Island of Mindoro are studied taxonomically. Five species of Hydraena (Hydraenopsis) are recorded of which three new species, namely H. hinundungan sp. n., H. quirao sp. n., and H. sanvicentensis sp. n., are described. Their genital characters are illustrated by line drawings. Photographs of their habitus and type localities, and a map of the collection sites are provided. Their habitat requirements are briefly discussed and their potential usefulness as bioindicators is deduced. The paper is based on a student thesis and intended as a first step towards the review of the entire hydraenid beetle fauna of Mindoro.

http://zoobank.org/urn:lsid:zoobank.org:pub:BDB0EABE-5A9A-46D0-83A2-81A3DA895BFD     

Cladispa Baly: revision,biology and reassignment of the genus to the tribe Spilophorini (Coleoptera: Chrysomelidae: Cassidinae)

The genus, Cladispa Baly 1858, is transferred from the tribe Imatidiini (= Cephaloleiini Chapuis, 1875) to Spilophorini Chapuis, 1875 based on the review of type material, newly collected specimens and molecular phylogenetic analysis. The type species, C. quadrimaculata Baly, 1858, is redescribed, and two new species, C. amboroensis sp.n. from Bolivia (Santa Cruz Department) and C. ecuadorica sp.n. from Ecuador (Pastaza Province), are described and figured. The morphology of C. amboroensis sp.n. immature stages is broadly consistent with other Spilophorini. Field observations document that both C. quadrimaculata and C. amboroensis sp.n. are trophic specialists on Orchideaceae. Keys to Cladispa species and Spilophorini genera are provided. Trophic associations of other Cassidinae and Orchideaceae are discussed. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:42A1ECF3‐2030‐4938‐8F3D‐FE7EC36F303A