Systematics and Biodiversity ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tsab20 Cryptic diversity in the mayfly Leptohyphodes inanis (Pictet) (Ephemeroptera: Leptohyphidae) across water basins in Southeastern Brazil Paula Malaquias Souto, Luiz Felipe Lima da Silveira, Daniela Maeda Takiya & Frederico Falcão Salles To cite this article: Paula Malaquias Souto, Luiz Felipe Lima da Silveira, Daniela Maeda Takiya & Frederico Falcão Salles (2021): Cryptic diversity in the mayfly Leptohyphodesinanis (Pictet) (Ephemeroptera: Leptohyphidae) across water basins in Southeastern Brazil, Systematics and Biodiversity, DOI: 10.1080/14772000.2021.1933248 To link to this article: https://doi.org/10.1080/14772000.2021.1933248 View supplementary material Published online: 21 Jun 2021. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tsab20 Systematics and Biodiversity (2021), 0(0):1–21 Research Article Cryptic diversity in the mayfly Leptohyphodes inanis (Pictet) (Ephemeroptera: Leptohyphidae) across water basins in Southeastern Brazil PAULA MALAQUIAS SOUTO1,2 , LUIZ FELIPE LIMA DA SILVEIRA3 , DANIELA MAEDA TAKIYA4 & FREDERICO FALCÃO SALLES5 1Programa de Pos-graduac ¸~ao em Biologia Animal (PPGBAN), Universidade Federal do Espırito Santo, Vitoria, ES 29075- 910, Brazil 2Linking Landscape, Environment, Agriculture and Food (LEAF), Tapada da Ajuda, 1349-017 Lisboa, Portugal 3Department of Biology, Western Carolina University, Cullowhee, NC 28723, USA 4Laboratorio de Entomologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-901, Brazil 5Museu de Entomologia, Departamento de Entomologia, Universidade Federal de Vic¸osa, Minas Gerais 36570-900, Brazil Leptohyphodes inanis (Pictet) is an enigmatic species with a rare trait among leptohyphid males – large and divided compound eyes. In addition, the color of its upper portion is variable across – but not within – populations. However, the geographic variation of this trait and its relation to gene flow across populations remain unknown. Here, we analyzed individuals across Southeastern Brazil (19 to 24S and 40 to 48 W) to (i) assess genetic (COI) and eye color variation, and (ii) evaluate if L. inanis is a single species, by combining Bayesian phylogenetic analyses (including two other leptohyphid genera – Tricorythopsis and Tricorythodes) and species delimitation methods: ABGD and mPTP. To further investigate within-species variation in phenotypic traits, we evaluated quantitative and qualitative morphological traits of 1,252 individuals. We found that genetic variation in L. inanis is largely unrelated to eye color, and that pairwise genetic divergences in COI mtDNA are remarkably higher (up to 30.7%) than previously found in other mayfly lineages. L. inanis was recovered as monophyletic, although results suggest it includes three to seven cryptic species, each one related to mountain ranges across Southeastern Brazil. Furthermore, we found no genetic variation among individuals of the same drainage basin, suggesting that populations might be largely isolated from one another. Because morphological traits traditionally used in Ephemeroptera taxonomy were ineffective in distinguishing the cryptic species, we propose L. inanis to be a species complex. Key words: Canastra mountain range, DNA Barcode, Mantiqueira system, phylogenetics, species delimitation Introduction implications for evolutionary theory, biogeography and Most descriptions and species delimitations of insect conservation planning (Bickford et al., 2007). taxa are based upon morphological characters alone, Different studies on genetic variation, often associat- which sometimes are inaccurate and render species iden- ing morphological and geographic data, revealed the tification a difficult or impossible task when dealing existence of cryptic species in several groups of animals with cryptic species (i.e., two or more distinct species and plants worldwide, in diverse types of habitats (e.g., morphologically indistinguishable and classified as a Dawson & Jacobs, 2001; Feulner et al., 2006;Gomez single species; Pfenninger & Schwenk, 2007). The study et al., 2002; Grundt et al., 2006; Hebert et al., 2004;de of cryptic species represents a challenge to taxonomists Rezende Dias et al., 2018; Vrijenhoek et al., 1994). The but is nevertheless crucial towards a comprehensive advent of molecular techniques and analyses has brought understanding of biodiversity patterns, and yields several about increased awareness of the importance of DNA data in insect taxonomy, which has proven very useful in the discovery of cryptic species (e.g., Bickford et al., Correspondence to: Paula Malaquias Souto. E-mail: 2007; Cardoni et al., 2015; Cook et al., 2008; Fujita [email protected] et al., 2012; Hebert et al., 2003; Hendrich et al., 2015; ISSN 1477-2000 print / 1478-0933 online # The Trustees of the Natural History Museum, London 2021. All Rights Reserved. https://dx.doi.org/10.1080/14772000.2021.1933248 Published online 21 Jun 2021 2 P. M. Souto et al. Figs. 1 and 2. L. inanis, nymph (a) from Santa Teresa (Espırito Santo) and male subimago (b) from Peixe Tolo river (Serra do Intendente State Park, Minas Gerais). Photos by Frederico Salles. Macher et al., 2016; Ossa-Lopez et al., 2017; Petit & rarer feature in Ephemeroptera: variation in the colour Excoffier, 2009; Silveira et al., 2016). of the upper portion of the males’ eyes. Insects are one of the most well represented groups in Although type-specimens of L. inanis have black the cryptic species literature, being the description and eyes, we have found that different populations show recognition of them of great implication for human variable coloration of the upper portion of compound health (e.g., Anopheles malaria-transmitting mosquitoes), eyes, in shades of red or black, followed by no other pest management (different species have variable pesti- obvious morphological distinction. In this context, the cide resistance), and studies of coevolution and species objective of the present work was to investigate whether interaction (Bickford et al., 2007). More recently, sev- eye coloration variation could be used to diagnose eral studies using molecular tools have drawn attention mtDNA lineages of L. inanis. Our findings suggest that to the existence of cryptic species complexes in L. inanis is a species complex, with each cryptic species Ephemeroptera (e.g., Gill et al., 2016; Macher et al., mostly restricted to different mountain ranges in 2016; Ossa-Lopez et al., 2017; Pereira-da-Conceicoa Southeastern Brazil. Although eye coloration couldn’t et al., 2012; Rutschmann et al., 2014; Williams et al., distinguish cryptic species alone, as different cryptic 2006), especially using information from the mtDNA species may have the same eye colour, it is a stable cytochrome C oxidase subunit I (COI) gene. However, character within each of the delimited species. Due to the within-species genetic variation remains largely the newly discovered cross-population variation in male unexplored in South American mayflies. eye colour, we also propose an expansion of the gen- Leptohyphodes Ulmer, 1920 (Ephemerelloidea: eric diagnosis. Leptohyphidae) was established for eight male imagos from Brazil, described as “Potamanthus ? inanis” by Pictet (1843) (Ulmer, 1920). Ulmer (1921) placed in Material and methods this genus another enigmatic taxon, Tricorythus australis Banks (1913), transferred later to Tricorythodes by Sampling, morphology, and taxonomy Traver (1958) and, more recently, to Macunahyphes Material examined is deposited at: (DZRJ) – Colec¸~ao Dias, Salles & Molineri (2005). The most unique char- Entomologica Prof. Jose Alfredo Pinheiro Dutra, acter of Leptohyphodes is a rare trait among leptohyphid Departamento de Zoologia, Instituto de Biologia, males: the large and divided compound eyes. This fea- Universidade Federal do Rio de Janeiro, Rio de Janeiro, ture is shared only with Amanahyphes Salles & Brazil; (CZNC) – Colec¸~ao Zoologica Norte Capixaba, Molineri, 2006 and Leptohyphes populus Allen (1973), Universidade Federal do Espırito Santo, S~ao Mateus, a species known only from a male nymph from Brazil; and (MZUSP) – Museu de Zoologia, Amazonas State (Brazil). Thus, Leptohyphodes is a Universidade de S~ao Paulo, S~ao Paulo, Brazil. Syntypes monotypic genus endemic to Southeastern Brazil. Its of L. inanis, deposited at Naturhistorisches Museum type species, Leptohyphodes inanis (Pictet, 1843) (Figs. Wien, Vienna, Austria (NMW), were observed by pho- 1 and 2), known from all the stages, also has an even tographs (courtesy of Dr. Ernst Bauernfeind, NHM). Cryptic diversity in the mayfly Leptohyphodes inanis 3 Material studied was collected in Southeastern Brazil, L. inanis, Tricorythodes eduardoi (Almeida & Mariano, in the states of Espırito Santo, Minas Gerais, Rio de 2015), Tricorythopsis chiriguano Molineri, 2001, Janeiro, and S~ao Paulo, mostly in the domain of the Tricorythopsis gibbus (Allen, 1967), and Tricorythopsis tropical Atlantic Rainforest, but some from Brazilian spongicola Lima, Salles & Pinheiro, 2011 Cerrado as well. The sampled area comprises three (Supplementary material Table S1). We used the mountain ranges: Serra do Mar, Serra da Mantiqueira, DNeasy Blood and Tissue Kit (Qiagen, Hilden, and Serra do Espinhac¸o. Our study area spanned about Germany) following a modified