Zoosyst. Evol. 95 (1) 2019, 1–13 | DOI 10.3897/zse.95.29487 A new species of Bungona in Turkey (Ephemeroptera, Baetidae): an unexpected biogeographic pattern within a pantropical complex of mayflies Pavel Sroka1, Roman J. Godunko1,2, Sereina Rutschmann3,4, Kamila B. Angeli5,6, Frederico F. Salles7, Jean-Luc Gattolliat8,9 1 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Branišovská 31, 37005 České Budějovice, Czech Republic 2 State Museum of Natural History, National Academy of Sciences of Ukraine, Teatralna 18, 79008 Lviv, Ukraine 3 Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310 Vigo, Spain 4 Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany 5 Laboratório de Sistemática e Ecologia de Insetos, Universidade Federal do Espírito Santo, Departamento de Ciências Agrárias e Biológicas, 29933-415, São Mateus, ES, Brazil 6 Programa de Pós-Graduação em Biologia Animal, Universidade Federal do Espírito Santo, 29075-910, Vitória, ES, Brazil 7 Museu de Entomologia, Departamento de Entomologia, Universidade Federal de Viçosa, 36.570-900, Minas Gerais, CEP, Brazil 8 Department of Ecology and Evolution, Biophore, University of Lausanne, 1015 Lausanne, Switzerland 9 Museum of Zoology, Palais de Rumine, Place de la Riponne 6, 1014 Lausanne, Switzerland http://zoobank.org/AFB9C305-2295-4B31-8667-2A2E1ACC2385 Corresponding author: Jean-Luc Gattolliat ([email protected]) Abstract Received 3 September 2018 By using an integrative approach, we describe a new species of mayfly, Bungona Accepted 4 January 2019 (Chopralla) pontica sp. n., from Turkey. The discovery of a representative of the trop- Published 25 January 2019 ical mayfly genus Bungona in the Middle East is rather unexpected. The new species shows all the main morphological characters of the subgenus Chopralla, which has its Academic editor: closest related species occurring in southeastern Asia. Barcoding clearly indicated that Martin Husemann the new species represents an independent lineage isolated for a very long time from other members of the complex. The claw is equipped with two rows of three or four flat- Key Words tened denticles. This condition is a unique feature of Bungona (Chopralla) pontica sp. n. among West Palaearctic mayfly species. Within the subgenus Chopralla, the species Biogeography can be identified by the presence of a simple, not bifid right prostheca (also present only Cloeodes complex in Bungona (Chopralla) liebenauae (Soldán, Braasch & Muu, 1987)), the shape of the Chopralla labial palp, and the absence of protuberances on pronotum. integrative taxonomy Middle East new species Introduction mayflies, a large proportion of species exhibits a broad Palaearctic or West Palaearctic distribution. A large part The Middle East is regarded as a transitory region con- of the fauna is composed of European species with rang- taining elements of Western and Eastern Palaearctic es extending into the Middle East (see Bauernfeind and faunas (Heller 2007). This explains why also the may- Soldán 2012 for distributional ranges of all European fly fauna of Turkey is very diversified, comprising 157 species). Another major group represents strictly Middle species reported in the most recent checklist (Salur et al. East species, including those described and known from 2016, with some later comments by Kazancı and Türk- only Turkey. Caucasian species also frequently occur, men 2016). Regarding biogeographic patterns of Turkish particularly in Eastern Turkey (Kazancı 2009, Türkmen Copyright Sroka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits un- restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Sroka et al.: New species of Bungona with unexpected distribution and Kazancı 2013). However, mayfly taxa usually show- genera Crassabwa Lugo-Ortiz & McCafferty, 1996, Che- ing preferences for tropical regions but occur in Turkey leocloeon Wuillot & Gillies, 1993, Dabulamanzia Lugo-Or- outside the tropical realm (and in the Middle East in gen- tiz & McCafferty, 1996 and Centroptilum Eaton, 1869. eral) at the same time are extremely rare. A part of the sequences used for the analysis were ob- In the present contribution, we describe such a taxon tained from the public databases, the rest was newly se- occurring in the Anatolian Peninsula, specifically a new quenced within the framework of the present study (see species from the genus Bungona Harker, 1957, subge- Table 1 for details including sources of data and acces- nus Chopralla Waltz & McCafferty, 1987. This subgenus sion numbers). We also searched in BOLD and GenBank currently encompasses seven species and is distributed databases for other existing similar sequences. However, in Borneo, Vietnam, Hong Kong, and Sri Lanka (Mar- no sequences more similar than 84% (and therefore po- le et al. 2016). Bungona is one of the four genera of the tentially informative for the analysis) were found except Cloeodes-complex (sensu Salles et al. 2016), this com- those already included in the dataset. plex containing Cloeodes Traver, 1938, Potamocloeon Voucher specimens of the new species, as specified in Gillies, 1990, and Crassolus Salles, Gattolliat & Sarto- Results, is deposited in the collection of the Biology Cen- ri, 2016. This complex is distributed in the Neotropical, tre of the Czech Academy of Sciences, Institute of En- Nearctic, Afrotropical, Oriental, and Australasian regions tomology, České Budějovice, Czech Republic (IE CAS: but is most diversified in the Pantropical area. Only a few holotype larva, three paratype larvae) and in the Musée species are reported from the Nearctic (Waltz and McCaf- cantonal de zoologie, Lausanne, Switzerland (MZL: two ferty 1987) and none from the Palaearctic. Consequently, paratype larvae). the discovery of this lineage in Asia Minor is extremely surprising, and thus, we discuss the relationships of the Morphological study new species with other members of the Cloeodes-complex Two paratype specimens of Bungona (Chopralla) pon- together with the evolutionary and biogeographic impli- tica sp. n. were mounted on slides, using HydroMatrix cations of this discovery. To achieve this goal, we use an (MicroTech Lab, Graz, Austria), Canada balsam, or Eu- integrative approach combining evaluation of morpholog- paral. Drawings were made using an Olympus SZX7 ste- ical characteristics with a molecular analysis of mtDNA reomicroscope and an Olympus BX41 microscope, both sequences. We also provide information on the evolution- equipped with a drawing attachment (camera lucida). ary history of the new species and a review of tropical Photographs were made using a Leica M205 C stereo- mayfly lineages reported from the Middle East up to now. microscope with a Canon EOS 1200D digital camera. All photographs were subsequently enhanced using Ado- be Photoshop CS5. For scanning electron microscopy Material and methods (SEM), the samples in 96% ethanol were transferred in three mixtures of graded series ethanol : acetone (2:1, 1:1 Taxon sampling and 1:2) with 15 minutes in each mixture and dried by A new species of mayflies Bungona (Chopralla) pontica means of a critical point dryer CPD 2 (Pelco TM). The sp. n. was collected in northern Turkey in 2011 (Fig. 8). samples were mounted on an aluminium target by means Herein, we describe the new taxon including both, mor- of carbon tape, coated with gold using a sputter coater phological and molecular techniques. To analyze the af- Baltec SCD 050. Finally, the samples were examined in finities of the new species based on the molecular data, a FE-SEM JSM 7401-F scanning electron microscope we built a dataset comprising sequences of several mayfly (JEOL Ltd, Tokyo, Japan) at low accelerating voltage of taxa, as summarized in Table 1. 4 kV using GB-low mode. The accomplished topographic In the selection of taxa, we wanted to include all major images provided a resolution of 1.5 nm by an efficient clades within the Cloeodes-complex, as inferred based on semi-in-lens detector. The morphological terminology morphological data by Salles et al. (2016). Specifically, the follows Salles et al. (2016). sampling included the genus Potamocloeon Gillies, 1990 with the subgenera Potamocloeon and Aquaediva Salles et Molecular study al. 2016, which together constitute a monophyletic group DNA extraction, amplification and sequencing.Genom - sister to all the rest of the Cloeodes-complex. We also in- ic DNA was extracted from whole specimens or legs us- cluded the genus Crassolus Salles et al. 2016, forming a ing the NucleoSpin 96 (Macherey-Nagel, Düren, Germa- sister lineage to the clade comprising the genera Cloeodes ny) tissue kit, the BioSprint 96 extraction robot (Qiagen), and Bungona. These latter two taxa were also included in or the Wizard SV Genomic DNA Purification System Kit the analysis, in case of the genus Bungona, all three subge- (Promega, California, USA), following the manufactur- nera (Bungona, Centroptella Braasch & Soldán, 1980 and er's protocol. We analyzed the 658 bp fragment of the mi- Chopralla Waltz & McCafferty, 1987) were sampled. There- tochondrial DNA barcoding gene cytochrome c oxidase fore, we achieved a comprehensive set of sequences from all subunit 1 (cox1). Standard polymerase chain reaction major phylogenetic lineages within the Cloeodes-complex. (PCR) amplifications were performed