www.nature.com/scientificreports OPEN Multilevel fne-scale diversity challenges the ‘cryptic species’ concept Received: 24 September 2018 Tatiana Korshunova 1,2, Bernard Picton 3,4, Giulia Furfaro 5, Paolo Mariottini 5, Accepted: 29 March 2019 Miquel Pontes 6, Jakov Prkić 7, Karin Fletcher 8, Klas Malmberg 9, Kennet Lundin 10,11 Published: xx xx xxxx & Alexander Martynov 2 ‘Cryptic’ species are an emerging biological problem that is broadly discussed in the present study. Recently, a cryptic species defnition was suggested for those species which manifest low morphological, but considerable genetic, disparity. As a case study we present unique material from a charismatic group of nudibranch molluscs of the genus Trinchesia from European waters to reveal three new species and demonstrate that they show a dual nature: on one hand, they can be considered a ‘cryptic’ species complex due to their overall similarity, but on the other hand, stable morphological diferences as well as molecular diferences are demonstrated for every species in that complex. Thus, this species complex can equally be named ‘cryptic’, ‘pseudocryptic’ or ‘non-cryptic’. We also present evidence for an extremely rapid speciation rate in this species complex and link the species problem with epigenetics. Available metazoan-wide data, which are broadly discussed in the present study, show the unsuitability of a ‘cryptic’ species concept because the degree of crypticity represents a continuum when a fner multilevel morphological and molecular scale is applied to uncover more narrowly defned species making the ‘cryptic’ addition to ‘species’ redundant. Morphological and molecular methods should be applied in concordance to form a fne-scale multilevel taxonomic framework, and not necessarily implying only an a posteriori transformation of exclusively molecular- based ‘cryptic’ species into morphologically-defned ‘pseudocryptic’ ones. Implications of the present study have importance for many felds, including conservation biology and fne-scale biodiversity assessments. Te ‘cryptic species’ concept is widely used in modern biodiversity studies1 and implies that there are morpho- logically indistinguishable species that can be recognized only by molecular data2,3. Te term ‘cryptic species’ became popular relatively recently4,5 and has supplanted the term ‘sibling species’ which was commonly used in previous cases with difcult-to-distinguish species6. Despite the fact that the ‘cryptic species’ concept has received considerable attention and is now used in various applications, it is intrinsically contradictory and might also be confused with other uses of the word ‘cryptic’. For example, the exact same ‘cryptic species’ term is used in ecology to denote that some species are very well camoufaged on some substrates7. Recently, the difculty of delineating the ‘cryptic species’ concept from the basic biological species defnition was highlighted, and it was suggested that this concept should be used with care and only as a temporary formal- ization for taxonomic complexes for which a robust morphological framework is not yet established8. In a further discussion, Struck et al.9 rather disagreed and attempted to build a defnition of ‘cryptic’ species on a lower degree of phenotypic (morphological) disparity than non-cryptic relatives. However, to defne an exact degree of mor- phological disparity is very difcult, if it is possible at all, so Heethof10 further concluded that the current ‘cryptic 1Koltzov Institute of Developmental Biology RAS, 26 Vavilova Str., 119334, Moscow, Russia. 2Zoological Museum, Moscow State University, Bolshaya Nikitskaya Str. 6, 125009, Moscow, Russia. 3National Museums Northern Ireland, Holywood, Northern Ireland, BT18 0EU, United Kingdom. 4Queen’s University, Belfast, Northern Ireland, United Kingdom. 5Department of Science, University of “Roma Tre”, Viale G. Marconi 446, I-00146, Rome, Italy. 6VIMAR - Vida Marina, Barcelona, Spain. 7Getaldiceva 11, C 21000, Split, Croatia. 8Port Orchard, Washington, 98366, USA. 9Aquatilis, Nostravägen 11, S-41743, Gothenburg, Sweden. 10Gothenburg Natural History Museum, Box 7283, SE- 40235, Gothenburg, Sweden. 11Gothenburg Global Biodiversity Centre, Box 461, SE-40530, Gothenburg, Sweden. Correspondence and requests for materials should be addressed to A.M. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:6732 | https://doi.org/10.1038/s41598-019-42297-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports species’ concept represents either conceptual or terminological chaos. Additional complications arise from the most recent practice in diferent animal groups such as crustaceans11, insects12 or echinoderms13 when the term ‘cryptic’ species is used for morphologically diagnosable species. Te ‘cryptic’ species problem is not merely a theoretical problem. Te strong trend to distinguish ‘cryptic’ and ‘non-cryptic’ species, and the explosive growth of exploration and description of ‘cryptic’ diversity, without a clearly defned terminological ground, is already resulting in recent outstanding controversies in important felds such as conservation biology - particularly regarding suggestions for special regulations of species names, that will afect the core of taxonomy14,15. Te term ‘cryptic species’ is also commonly vaguely applied, making its defnition uncertain10, which prompts the necessity of its revision. Te present case is of special importance since it links a taxonomic problem in a particular organism group with the most general biological problem of the species concept, using broad-scope material on marine animals. Here, we show that a very colourful species of nudibranch mollusc Trinchesia caerulea (Montagu, 1804), which was originally described from the UK, but common throughout European waters (including Norway, Sweden and the Mediterranean region), is revealed to actually be a complex of four species (three of which are new). According to the present study, these species are robustly distinguished by their molecular data and were never taxonomically separated previously and thus ft within the ‘cryptic species’ concept and its updated defnition including “low morphological disparity9”. However, the apparent cryptic morphology for that complex had previously been noted and questioned16,17 and in the pres- ent study those morphological distinctions have been supported by novel molecular data. Tus, these ‘cryptic’ species can be distinguished morphologically (and hence can be defned as ‘pseudo-cryptic’ species) not only a posteriori afer molecular study, as was commonly suggested and has become a notable modern tendency18,19, but by using multilevel morphological information as a primary source. Tis adds a new perspective to the under- standing of the core biological species problem, which should not just be to adhere to a putative cryptic notion, but methods should rather follow the idea “from cryptic to obvious species” with the rapid progress of various molecular analyses and aids to morphological diferentiation20,21 in order to reveal an immense hidden multilevel biological diversity. Materials and Methods Material for this study was obtained by scuba diving at widely separated locations across Europe: from the United Kingdom, Ireland, Norway, Sweden, Spain, Italy, France, Croatia and Russia. Te specimens were deposited in the Zoological Museum of Lomonosov Moscow State University (ZMMU), in the Gothenburg Natural History Museum (GNM), National Museums Northern Ireland (BELUM.Mn), and the Department of Science of the Roma Tre University (RM3). Integration of molecular and morphological data as well as phylogenetic and biogeographical patterns were used. Te external and internal morphology of the 28 spec- imens was studied using digital cameras, under a stereomicroscope and scanning electron microscope. For molecular analysis 17 specimens were successfully sequenced for the mitochondrial genes cytochrome c oxi- dase subunit I (COI), 16S rRNA, and the nuclear genes Histone 3 (H3). Te DNA extraction procedure, PCR amplifcation options, and sequence gathering are described in detail in previous studies8,22–25. Additional molecular data for 13 specimens of nudibranchs were obtained from GenBank (see supplementary informa- tion Table S1). Outgroup selection was based on previous studies23,25–27. Two diferent phylogenetic methods, Bayesian Inference (BI) and Maximum Likelihood (ML), were used to infer evolutionary relationships. To evaluate the genetic distribution of the diferent haplotypes, a haplotype network for the COI molecular data was reconstructed using Population Analysis with Reticulate Trees (PopART, http://popart.otago.ac.nz). Also, the minimum uncorrected p-distances between all the sequences as well as maximum intra- and minimum intergroup genetic distances were examined. Automatic Barcode Gap Discovery (ABGD)28 was used to defne species. See supplementary information Text for methods in detail. Results Molecular phylogenetic relationships and morphological data of a nudibranch species complex. Phylogenetic analysis was performed using 28 specimens of Trinchesia, including data for 21 Trinchesia caerulea species complex specimens, four other congener species of the genus Trinchesia and two outgroup species. All supposedly morphologically cryptic morphs of Trinchesia caerulea illustrated in Tompson & Brown (1984)16 were collected in European waters and examined. In addition, specimens collected in the Black Sea that are taxonomically close to T. caerulea were