Organisms Diversity & Evolution (2018) 18:291–312 https://doi.org/10.1007/s13127-018-0374-6 ORIGINAL ARTICLE Two new bioluminescent Henlea from Siberia and lack of molecular support for Hepatogaster (Annelida, Clitellata, Enchytraeidae) Emilia Rota1 & Svante Martinsson2 & Christer Erséus2 Received: 25 February 2018 /Accepted: 30 July 2018 /Published online: 30 August 2018 # Gesellschaft für Biologische Systematik 2018 Abstract Two bioluminescent enchytraeids, Henlea petushkovi sp. n. and Henlea rodionovae sp. n., are described from the Krasnoyarsk and Irkutsk regions in Eastern Siberia. These large potworms exhibit the typical light-production pattern reported repeatedly in the genus and recently elucidated by Russian researchers in its main biophysical and biochemical aspects. Morphology and DNA indicate that the two species are very closely related, but clearly divergent in the strength of the body wall (thick and opaque in H. petushkovi), structure of the prostomium (in H. rodionovae unprecedentedly wrinkled and mobile), brain shape (almost equilat- eral in H. petushkovi), size of coelomocytes (60–85 μminH. petushkovi) and structure of intestinal diverticula (tulip-shaped in H. petushkovi, apple-shaped in H. rodionovae). Limited hybridization seems to occur between them, supported by a single case of conflict between COI and morphology, and a few intermediate morphotypes were noted in greenhouse populations. The new species are phylogenetically distant from all known congeners so far DNA-barcoded, even those that, like them, respond to the diagnosis of the putative subgenus Hepatogaster Čejka, 1910 (multitubular gut diverticula in VIII, indented brain, dorsal blood vessel from IX, prominent spermathecal glands, and nephridia from 5/6). In fact, our phylogenetic analyses dismiss Hepatogaster as an artificial (polyphyletic) taxon. Issues related to the definition of H. nasuta (Eisen, 1878), H. ochracea (Eisen, 1878) and H. irkutensis Burov, 1929, three species originally described from Siberia, indicate that Henlea taxonomy is still in a state of flux, as regards not only species interrelationships but also species definitions. Keywords New species . Henlea . Taxonomy . Cytochrome C oxidase subunit 1 . Histone 3 . Hybridization . Phylogeny . Hepatogaster . Bioluminescence Introduction technology (by Valentin N. Petushkov and Natalja S. Rodionova since 2002, 2003; see references in Rodionova et Light production in the Enchytraeidae has been known since al. 2017). It was the discovery of Fridericia heliota Zalesskaja the nineteenth century (see historical reviews in Rota et al. (in Zalesskaja et al. 1990), a brightly glowing Siberian forest 2003;Rota2009), but it is only during the last two decades dweller, that sparked new interest and a rigorous effort to- that the issue has been approached with modern insight and wards understanding the mechanism as well as the distribution of the phenomenon across the family. Light production had never been recorded in Fridericia Michaelsen, 1889 before, Electronic supplementary material The online version of this article and in such an amazing form (a continuous, bright glow of the (https://doi.org/10.1007/s13127-018-0374-6) contains supplementary body wall) as in F. heliota. All previous accounts of material, which is available to authorized users. enchytraeid luminescence reported flashes of light by worms identified either as Enchytraeus Henle, 1837 or as Henlea * Emilia Rota [email protected] Michaelsen, 1889 species. Therefore, it was also of great im- portance to compare the tissue origin and chemical bases of light emission in the different genera. It should be said upfront 1 Department of Physics, Earth and Environmental Sciences, University of Siena, Via P.A. Mattioli 4, 53100 Siena, Italy that the phenomenon in the genus Enchytraeus as currently conceived is not confirmed, thus, early reports under that 2 Systematics and Biodiversity, Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, name (e.g., Owsiannikow 1864;Harker1887; Pütter 1905) SE-40530 Göteborg, Sweden were possibly all misnamed records of Henlea. 292 Rota E. et al. Light production in Henlea was described in detail by specimens were found in taiga soil along Kacha and Mana Walter (1909) from specimens collected in garden soil of rivers in the region of Krasnoyarsk, together with F. heliota, Kaluga and Perm districts, European Russia, both identified but later, the same or closely related luminous worms were by Wilhelm Michaelsen as Henlea ventriculosa (d’Udekem, detected in other places, outdoor or more frequently in green- 1854). Luminosity was subject to fluctuations and always houses, and as far as Lake Bajkal. Beside the different body appeared more intense at the body ends, and accompanied localization, the bioluminescence system of the Henlea by discharge of luminous slime upon mechanical, chemical worms shows, with respect to F. heliota, different spectral or thermal stimulation. In the same period, similarly luminous characteristics and different temperature and pH optima, and enchytraeids were found in Austrian greenhouses by botanists its distinctness is confirmed by the negative results of all pos- Linsbauer (1917) and Molisch (1912). Linsbauer considered sible cross-reactions. Biochemically, the system of Henlea the animals self-illuminating, not having been able to detect comprises four essential components: luciferase, luciferin, ox- bacteria within the worms or their mucus (Pratje 1923). ygen and calcium ions. For F.heliota, the luminescent reaction Recently, during studies of F. heliota, some large Siberian requires five components: luciferase, luciferin, ATP, magne- Henlea worms (Fig. 1) were noted to exhibit the same pattern sium ions and oxygen (see Rodionova et al. 2017). of bioluminescence as described by Walter (1909)and A taxonomic description of these bioluminescent Siberian Linsbauer (1917), in response to tactile or chemical distur- Henlea worms, which are in fact different from H. ventriculosa bance (Petushkov et al. 2002; Rota et al. 2003). The first and comprise an evolutionary lineage with considerable genet- ic variation, is given here. What we consider to be two closely related, but morphologically divergent, bioluminescent species in this lineage, H. petushkovi sp.n.andH. rodionovae sp. n., are present both in the region of Krasnoyarsk and around the Bajkal. However, because they appear to be interfertile, and a few morphological intermediates were observed in samples from greenhouses in these regions, they will be described here from the most divergent morphotypes collected. On Hepatogaster Čejka, 1910 Both our two new species fit the diagnosis of Hepatogaster Čejka, 1910, a taxon considered by Welch (1920)and Stephenson (1930) subordinate to the genus Henlea, but that Černosvitov (1937) maintained in a distinct position within the Henleinae. The species included in Hepatogaster by Černosvitov (1937) indeed satisfy the original uncommon criteria established by Čejka (1910): (1) oesophageal append- ages extending into VI and VII, (2) “gastrointestinal glands”, forming a quadruple multitubular structure surrounding gut in VIII, (3) spermathecae opening into the oesophagus in the very back of V (in VI from an external view), (4) a gradual dilatation of the oesophagus to form the intestine. What prin- cipally differentiates the named (sub) group from the other Henlea species also possessing oesophageal diverticula in VIII and dorsal vessel originating in IX (H. ventriculosa, H. jutlandica Nielsen and Christensen 1959), is the multitubular substructure (as opposed to a largely hollowed cavity) of the pouches closely surrounding the gut in VIII. Figure 2 shows how Michaelsen (1886) illustrated through histological cross-sections the different structures of the gut diverticula in Enchytraeus leptodera Vejdovský 1879 (= Henlea nasuta auct.) and E. ventriculosus d’Udekem (= H. ventriculosa). In E. leptodera, two lateral pouches protrude Fig. 1 Bioluminescent Henlea specimen photographed in vivo. a Under a light microscope. b By direct contact printing worm-to-film in the dark from the intestine and project freely forward into the body- room (courtesy of V. N. Petushkov & N. S. Rodionova). Scale bar: 1 mm cavity (Fig. 2a). In E. ventriculosus, four pockets closely Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 293 Fig. 2 Histological cross sections of the gut diverticula in Enchytraeus leptodera Vejdovský (a)andEnchytraeus ventriculosus d’Udekem (b), and in Archienchytraeus nasutus Eisen (c), as shown by Michaelsen (1886) and Michaelsen (1889), respectively adhere to the intestine (Fig. 2b). In both cases, the lumen of the The status of Hepatogaster was briefly commented upon in pockets is constricted by the multiple, irregular folds of the morphocladistic terms by Tynen et al. (1991). In this paper we walls, so that a considerable enlargement of the inner surface will attempt to verify if this old name deserves taxonomic takes place. Later, Michaelsen (1889) illustrated the structure status, or not, on a molecular basis. of the gut diverticula in the types of Archienchytraeus nasutus Eisen, 1878, where two short, wide pockets are attached lat- Taxonomy and diversity of Henlea in Siberia erally to the posterior end of the oesophagus, and extend for- ward without fusing with it. The walls of the pockets are Taxonomic work on the enchytraeids of Siberia has mostly “often and irregularly folded, and so strongly that the cen- concerned