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2015 De Freina Et Al Chondrostega Escobesae-1.Pdf J J. F et al., Notes on Chondrostega in the Iberian Peninsula Notes on the biology, distribution and taxonomy of Chondrostega L, 1857 in the Iberian Peninsula with a description of the southern Spanish Chondrostega escobesae sp. nov. (Lepidoptera: Lasiocampidae, Chondrosteginae) ●J J. F, Y M L­, C A A & R V Abstract. Spanish populations of Chon- Resumen. Consideraciones sobre la Zusammenfassung. Von spanischen drostega L, 1857 are examined biología, distribución y taxonomía de Populationen der Gattung Chondrostega based on external and internal morphol- Chondrostega L, 1857 en la pe- L, 1857 werden äußere und inne- ogy as well as molecular data from the nínsula ibérica, con la descripción de re morphologische Merkmale wie auch mitochondrial cytochrome oxidase sub- Chondrostega escobesae sp. nov. del molekulare Datensätze der mitochond- unit I gene (COI). According to relevant sur de España (Lepidoptera: Lasio- rial cytochrome oxidase subunit I gene diagnostic features and genetic differen- campidae, Chondrosteginae) (COI) untersucht und ausgewertet. Die tiation, two species with allopatric dis- Se realiza una revisión taxonómica de las Diagnose belegt die Existenz zweier al- tribution are recognized within the Ibe- poblaciones españolas de Chondrostega lopatrisch wie auch genetisch getrennter rian Peninsula. Andalusian populations, L, 1857, a partir del estudio de Arten auf der Iberischen Halbinsel. Po- here described as Chondrostega escobesae caracteres morfológicos internos y exter- pulationen Andalusiens werden als sp. nov., are compared to the closely re- nos, así como del gen mitocondrial cito- Chondrostega escobesae sp. nov. beschrie- lated Chondrostega vandalicia (M, cromo oxidasa subunidad I (COI). Aten- ben und mit ihrer Schwesterart Chond- 1865), which is limited to the northern diendo a características diagnósticas rostega vandalicia (M, 1865) ver- half of the Iberian Peninsula. They can relevantes, el género se divide en dos glichen, deren Verbreitung sich auf die be recognized by largely constant mor- especies alopátridas en la península ibé- Nordhälfte der Iberischen Halbinsel be- phological features such as a different rica. Las poblaciones presentes en Anda- schränkt. Beide Arten sind anhand kon- pattern on the underside of the wings of lucía se describen aquí como Chondros- stanter morphologischer Unterschiede the male, adult male genital structure, tega escobesae sp. nov. y son comparadas wie der verschieden ausgeprägten Hin- different structure of the canthus and the con Chondrostega vandalicia (M, terflügelzeichnung, der männlichen Ge- colour of hairs in the final instars. Phy- 1865), limitada a la mitad norte de Ibe- nitalstruktur, der Form des Canthus und logenetic analyses based on COI identify ria. Estas especies pueden ser diferencia- der deutlich voneinander abweichenden the two species as sister clades with a das a partir de varias características Färbung erwachsener Raupen unter- minimum genetic divergence of 3.8 %. como la existencia de un patrón diferen- scheidbar. Die Auswertung der DNA- Adults, including the apterous females, te en el reverso de las alas de los machos, Barcoding ergibt einen DNA-Sequenz- immature stages, larval host plants and la estructura genital masculina, diferen- unterschied des mtDNA Cytochromoxy- natural habitat are described and illus- te estructura del canthus y la coloración dase-I-Gens (COI) zwischen nord- und trated. A neotype is designated for C. de la pilosidad de los últimos estadios südspanischen Tieren von mindestens vandalicia. larvarios. El análisis filogenético basado 3,8 %. Der Beitrag liefert Beschreibungen en el COI muestra las dos especies como und Abbildungen der Imagines ein- clados hermanos con una divergencia schließlich des apteren Weibchens, der genética del 3.8 %. Se describen e ilus- letzten Präimaginalstadien, Wirtspflan- tran los adultos de ambos sexos, estadios zen und Lebensräume. Für C. vandalicia inmaduros, plantas nutricias de las oru- wird ein Neotypus designiert. gas y varios hábitats. Se designa además un neotipo para C. vandalicia. Key words. Lepidoptera, Lasiocampoidea, Lasiocampidae, Chondrosteginae, Chondrostega, Iberian Peninsula, new species, neotype, taxonomy, Palaearctic region, DNA barcoding, mitochondrial DNA. Introduction Chondrostega L, 1857 is a complex western Asia, whereas Chondrostegoides persed populations. This is especially true of species and forms, rather difficult to A, 1905 is limited to southern for the Chondrostega vandalicia (M, classify, that is distributed from the south- Africa. Theaptery of Chondrostega females 1865) species group, one of the cases with ern Europe and North Africa to central and has presumably resulted in many dis- controversial taxonomy in this genus 195 Entomologische Zeitschrift · Schwanfeld · ( (R V 1978, F the adults. Based on these morphological preserved in absolute ethanol. Total W 1987, L 2006), which displays differences and mitochondrial DNA re- genomic DNA was extracted using Chelex scattered populations from the Iberian sults, we propose that C. vandalicia exists 100 resin, 100–200 mesh, sodium form Peninsula and over the entire Maghreb to in the northern half of the Iberian Penin- (Biorad), under the following protocol: the Levant. Absence of diagnostic charac- sula, while the Andalusian populations the tissue was introduced into 100 μL of ters both in genitalia and larval stages has represent a separate species, here de- Chelex 10 % and 5 μL of Proteinase K led to descriptions of a considerable num- scribed as Chondrostega escobesae sp. nov. (20 mg⁄mL) were added. The samples ber of taxa of uncertain validity, listed were incubated overnight at 55 °C and below: were subsequently incubated at 100 °C for Methods and materials 15 minutes. Samples were then centri- C. constantina A, 1894; C. pow- Morphologie and repositories fuged for 10 seconds at 3.000 rpm. A 655- elli O, 1912; C. tingitana P , Cleaned, stained genitalia were stored and bp fragment at the 5’ end of the mitochon- 1916; C. maghrebica J, 1929 examined in 30 % ethanol, and slide- drial gene (COI) was amplified by poly- (described from populations of Morocco mounted in Euparal before being photo- merase chain reaction (PCR) using the and Algeria); C. zanoni T, 1922; C. graphed. primers UniLepF1 (5’-TAA TAC GAC TCA misuratana (K, 1939 (described CTA TAG GGA TTC AAC CAA TCA TAA from Libya); C. subfasciata K, 1830; C. Repository abbreviations are as follows: AGA TAT TGG AAC-3’) and UniLepR1 (5’- palaestrana S, 1891; C. fasciana BC – Barcode; CFM – Collection F, ATT AAC CCT CAC TAA AGT AAA CTT S, 1891; C. longespinata A- München; CML – Collection M CTG GAT GTC CAA AAA ATC A-3’), with , 1894; C. aurivillii P, 1902; L , Logroño; GPdF – Genitalia dissec- universal T7 and T3 tails here highlighted C. goetschmanni S , 1915; C. osthel deri tion F; IBEB – Institut de Biologia in bold.PCR was carried in 25-μL volumes P, 1925; C. intacta G, 1933; Evolutiva (CSIC-UPF), Barcelona; ID – containing: 14.4 μL autoclaved Milli-Q C. pauli G, 1933; C. schwingenschussi Identifying number; MWM – Collection of water, 5 μL 5x buffer, 2 μL 25 mM MgCl2, Z , 1933; C. elema W, 1941; Lepidopterological Museum W, Mu- 0.5 μL 10 mM dNTPs, 0.5 μL of each prim- C. brunneicornis W, 1944 (all de- nich; ZMHU – Museum für Naturkunde, er (10 μM), 0.1 μL Taq DNA Polymerase scribed from Levantine populations). Berlin (formerly Zoologisches Museum (Promega, 5U/μL) and 2 μL of extracted der Humoldt-Universität, Germany); ZSM DNA. The typical thermal cycling profile To test the validity of these taxa, further – Bavarian State Collection of Zoology, was: first denaturation at 92 °C for 60 s, research is essential and molecular data Munich. followed by five cycles of 92 °C for 15 s, may shed light on the extent of diversifica- 49 °C for 45 s and 62 °C for 150 s, and then tion among populations. It should be ex- Description and comparative diagnosis is by 35 cycles of 92 °C for 15 s, 52 °C for 45 s pressly mentioned here that due to lack of supplemented by biogeographical and and 62 °C for 150 s and a final extension at scientific verification we can not accept biological data concerning C. vandalicia 62 °C for 420 s. PCR products were purified the arbitrary taxonomic changes within and the new species C. escobesae. This in- and sequenced by Macrogen Inc. using the the Chondrostega group made by L formation has been obtained as a result of T7 and T3 universal primers. Sequences (2006). This means that we still consider intensive observations of immature stages were edited and aligned using GENEIOUS C. vandalicia as non-conspecific with any and life history both in natural conditions PRO 6.0.5 created by Biomatters (http:// of the North African taxa. and captive breeding by Y. M. L., C. A. A. www.geneious.com/). A Neighbour-Join- and further supporting lepidopterologists ing (NJ) phylogenetic tree was obtained The Iberian populations of Chondrostega (see acknowledgements). The study of the using MEGA 6 (T et al. 2013), based have all been previously treated as belong- life-cycle was carried out in several loca- on p-distance and pairwise deletion. Node ing to C. vandalicia (M, 1865). tions in Iberia over a period of several supports were assessed through 100 boot- However, we show that the genital struc- years to the present day. strap pseudo-replicates. Sequences for two ture, canthus morphology and DNA se- lasiocampid species were obtained from quence data of the southern Spanish pop- GenBank and used as outgroup. Genetic ulations display considerable differences DNA Analysis distances between species are reported as compared to those in northern Spain. We analyzed the cytochrome c oxidase I minimum uncorrected pairwise distances, Furthermore, morphological differences (COI) mitochondrial marker for ten Chon- while intraspecific variation is reported as in male adults, as well as a significant dif- drostega specimens representing five pop- maximum uncorrected pairwise distances. ference in larval colour, suggest that two ulations in the Iberian Peninsula. DNA was geographically separate species exist, de- extracted from one leg removed from DNA extracts are stored at the IBEB.
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