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, , )

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 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 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 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 material collected at the highest latitudes, in the tral cavity of the pockets almost completely recedes against Arctic tundra (Eisen 1878, 1879; Čejka 1910, 1912, 1914; the folds and their interstices” (Michaelsen 1889;Fig.2c). Nurminen 1980; Piper et al. 1982; Christensen and Dózsa- Welch (1920), Stephenson (1922, 1930)andČernosvitov Farkas 1999). Studies of the faunas of taiga and mixed forests (1931, 1937) speculated about an ontogenetic and phylo- are basically limited to collections in the Yenisej river basin, genetic transition between the different degrees of folding i.e., along the main river course down to Vorogovo (Eisen of the diverticula walls, from being fairly even and smooth 1879), and along its tributaries by the cities of Krasnoyarsk, (as seen in H. ventriculosa) to variously folded (as seen in Bratsk and Irkutsk (Burov 1929;Nurminen1973a;Rotaetal. H. nasuta) to the fusion between the adjacent folds to form 2003). On one occasion, samples were also taken from the the tubuliferous condition of Hepatogaster.Infact, vegetated shores of Lake Bajkal, south of Irkutsk (Nurminen Černosvitov (1931, 1937) placed Hg. sibiricus Čejka, 1973a). All these studies indicate Henlea sensu lato as one of 1910 outside Hepatogaster because of its incomplete re- the dominant enchytraeid genera in the region. At the species placement of folds by tubules. level, Nurminen (1973a) reconducted the recorded diversity to 294 Rota E. et al. six elements: four Henlea species widely distributed in the and whole-mounted in Canada balsam. The anterior region of Holarctic (H. heleotropha Stephenson, 1922, H. nasuta, H. some other worms was embedded in paraffin, sectioned either perpusilla Friend, 1911, H. ventriculosa), an augmented H. transversely or longitudinally at 7 μm and stained with ochracea (Eisen, 1878) (where the same author lumped six haematoxylin and eosin. Morphometric measurements refer Nordic species with multitubular diverticula (Nurminen to adult live specimens except where indicated, and were done 1973b); see taxonomic notes on this species in under an eyepiece micrometer. In text and figures, segments “Discussion”), and H. irkutensis Burov, 1929 (later renamed are referred to by Roman numerals; intersegmental furrows Punahenlea irkutensis; see more remarks in “Discussion”). and septa are referred to by Arabic numerals (fractions). Our study of luminous and non-luminous Siberian Henlea Chaetal formulae are indicated as follows: lateral preclitellar material adds to the body of evidence of a vast autochthonous − lateral postclitellar: ventral preclitellar − ventral postclitellar diversity of this genus in Siberia and suggests the necessity of (‘÷’ = from ... to ...); infrequent numbers are given in paren- reassessing some old species descriptions and subsequent theses. Photographs were taken with a DM LB Leica micro- classifications and synonymizations with European taxa. We scope connected to a Coolpix4500 Nikon digital camera. refer in particular to H. nasuta, H. ochracea and H. irkutensis, For genetic studies, live luminous and non-luminous three species originally described from Siberia, whose current- Henlea worms from greenhouses in Elovoye and Bolshie ly accepted definition poses questions concerning their true Koty were characterized morphologically, then fixed in 85% identities. No revision of types will be made here, but some ethanol; their caudalmost 20 segments immediately cut off nomenclatural issues will be discussed, as a preliminary con- and transferred to 96–100% ethanol, and later mailed to tribution to clarify interrelationships within Henlea. Sweden for DNA analysis. Type material of the two new species has been deposited in the Museo Civico di Zoologia di Roma (MCZR), Rome, and Material and methods the Swedish Museum of Natural History (SMNH), Stockholm.

Luminous Henlea worms were found in two Siberian regions, Abbreviations used in figures br, brain; cc, coelomocytes; co, located nearly 1000 km from one other. In the Krasnoyarsk collar of sperm funnel; di, gut diverticula; dv, dorsal vessel region, within a radius of 50 km of the city, and precisely: (1) origin; ed, ectal duct of spermatheca; end, ental duct of sper- taiga forest soil by Kacha river (56° 14′ 13.53″ N, 92°25′ 9.15″ matheca; gl, ectal glands of spermatheca; hp, head pore; i, E), immediately south of the Biological Station of Krasnoyarsk intestine; mp, male pore; ne, nephridium; oe, oesophagus; p, State University, together with F. he li ota; (2) taiga forest soil prostomium; pb, penial bulbs; phg, pharyngeal glands; pp, along Mana river (between 55°51′ 20.78″ N, 92°32′50.69″ E pharyngeal pad; sa, spermathecal ampulla; sf, sperm funnel; and 55°42′30.25”N, 92°55′14.72″E; habitat as described in sv, seminal vesicle; vd, vas deferens; vv, ventral vessel. Rota et al. 2003), together with F. heliota; (3) wooded yard of a farmhouse in Gladkoye, near Podolka (56°21′33.27”N, 92°26′7.51″E), together with F. heliota; (4) soil within and DNA-extraction, amplification and sequencing around greenhouse at Elovoye, near Krasnoyarsk airport (56°8′40.08”N, 92°32′27.97″E). In the Irkutsk region, near A first attempt at investigating the phylogenetic relationships the southern tip of Lake Bajkal, and precisely: (5) soil within of the bioluminescent Henlea lineage was made in 2014–15 and around greenhouse in Listvyanka (51°50′52.7”N, 104°52′ (by colleagues of Moscow State University), based on COI, 43.0″E), and (6) soil within and around greenhouse in Bolshie 12S, 16S, and 18S sequences from ten worms collected in Koty (51°54′23.5”N, 105°4′32.1″E). Specimens were extract- Gladkoye (Krasnoyarsk region). For our study (at University ed from soil samples by picking them out manually in a dark of Gothenburg) aimed to test whether the morphological var- room (with some minimal contamination by non-luminous iation observed in the bioluminescent lineage actually individuals). Self-luminous photographs (Fig. 1b) were taken reflected genetic divergence, we chose as molecular markers by direct contact printing worm-to-film through thin polyeth- the Cytochrome C Oxidase subunit I (COI) and Histone H3 ylene film in the dark room (using b/w SVEMA film ASA 100 (H3). Furthermore, to estimate phylogeny in a larger sample of and an exposure time of 1–5s). Henlea species, we also added the genes 12S, 16S, 18S, and Live Henlea specimens (luminous and non-luminous) col- 28S. DNA was extracted and amplified from 20 morphologi- lected by V.N. Petushkov and N.S. Rodionova from the above cally characterized Henlea specimens (of which 18 localities were shipped to the first author in Italy. The worms bioluminescent and 2 non-bioluminescent) collected in green- were first examined microscopically and re-checked for bio- houses in Elovoye (Krasnoyarsk region) and Bolshie Koty luminescence while alive, then fixed in 70–85% alcohol or in (Irkutsk region), using Epicentre’s QuickExtract DNA Bouin’s fluid. Some preserved specimens were stained in Extraction Solution 1.0, and the PCR primers and programs paracarmine and/or dehydrated in ethanol, cleared in xylene described in Martinsson et al. (2017). Attempts to amplify also Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 295 the Internal Transcribed Spacer 2 (ITS2) proved unsuccessful, HKY + Γ substitution model, empirical base frequencies, a due to length-variable heterozygosity. Sequencing was carried strict clock model with a rate of 1, the coalescent/constant size out by Eurofins MWG Operon (Ebersberg, Germany). tree prior; with constant population size prior set as exponential Sequences were assembled into consensus sequences using with a mean of 1.0. The analyses were run in BEAST v.1.8.2 Geneious v.7.1.8 (Biomatters Ltd., Auckland, New Zealand). (Drummond and Rambaut 2007; Drummond et al. 2012)for The bioluminescent specimens proved to belong to two new 10 million generations, sampling every 1000th generation. species (H. petushkovi and H. rodionovae), and the non- Tracer v. 1.6 (Rambaut et al. 2014) was used for examining bioluminescent Siberian specimens also represented two un- the effective sample size (ESS) for parameters and determining identified species, probably new, here denoted “sp. A” and the burn-in. Trees and posterior probabilities were summarized “sp. B”. Sequences from other Henlea species (H. glandulifera using TreeAnnotator v. 1.8.2 (Drummond and Rambaut 2007) Nurminen, 1970, H. groenlandica Černosvitov, 1929 augm. and showed on the Maximum clade credibility tree, discarding Christensen and Dózsa-Farkas 2006, H. heleotropha, H. the first 10% as burn-in. magnaampullacea Dózsa-Farkas et al., 2015, H. montana To visualize haplotype diversity, haplotype networks were Rota, 1994, H. nasuta, H. ochracea, H. perpusilla, H. constructed for COI and H3 genes in PopART v1 (Leigh and ventriculosa) and, as outgroups, Bryodrilus diverticulatus Bryant 2015) using statistical parsimony (Templeton et al. 1992; Černosvitov, 1929, Claparedrilus semifuscoides Klinth et al., Clement et al. 2002). We also calculated the uncorrected pairwise 2017b, Globulidrilus riparius (Bretscher, 1899), Marionina genetic distances (p-distances) for COI between the specimens communis Nielsen and Christensen, 1959, and Oconnorella of the two new species in MEGA 6 (Tamura et al. 2013). cambrensis (O’Connor, 1963), were either downloaded from Further, to find the intrageneric position of the two new GenBank, or in some cases newly generated for this study. It species, and to test if Hepatogaster forms a clade distinct from should be noted that H. magnaampullacea and H. ochracea the remaining Henlea species, a tree using six markers (COI, (like the two new species) morphologically correspond to the H3, 12S, 16S, 18S and 28S) combined and with a wider se- concept of Hepatogaster (see Table 1). lection of Henlea species was estimated, using Bayesian The sequences of each marker were aligned using the Inference in MrBayes v.3.2.6 (Ronquist et al. 2012). As there Geneious alignment with default settings in Geneious v. 7.1.8. are two paralogues of H3 in the bioluminescent species (see In the H3 dataset, all except one of the specimens of the two “Results”) only one of them, H3a, was used in this analysis. new species showed clear signs of heterozygosity, i.e., there The dataset was partitioned according to marker, and the pro- were distinct double peaks at certain positions in the sequencing tein coding genes COI and H3 were partitioned according to chromatograms, caused by the presence of two H3 paralogues codon position. The partitions were unlinked, rate variation (see “Results”). Due to this, the H3 paralogues were separated across sites was set to gamma distribution with a proportion using the PHASE algorithm (Stephens and Donnelly 2003; of invariable sites; model jumping was implemented to inte- Stephens et al. 2001) as implemented in DNAsp v.5.10 grate over substitution model space. The analysis ran for 10 (Librado and Rozas 2009), the phasing was run for 100 itera- million generations sampling every 10,000 generations. The tions after 100 initial burn-in iterations, with a thinning interval log files were inspected in Tracer v1.6 (Rambaut et al. 2014) of 1, using default settings. All sequences are deposited in for convergence and effective sample size. The first 25% of GenBank; see Table 1 for accession numbers, and details about the analysis was discarded as burn-in, and majority-rule con- the specimens used in the genetic analyses. A concatenated sensus trees were constructed. matrix including sequences from in total 13 species of AlltreesweredrawnwithFigTreev.1.4.1(Rambaut Henlea, and five outgroups was created: all included specimens 2014), and further edited in Adobe Illustrator CS5. had at least two of the six markers, i.e., CE27997 was excluded.

Molecular analyses Results

To test the genetic distinctness of H. petushkovi and H. Taxonomy and morphological descriptions rodionovae, single gene trees for COI and H3 were estimated by Bayesian coalescent analysis of the available sequences of Henlea petushkovi sp. n. (Figs. 3–5) these two new species (28S data did not show enough varia- tion, and were not used in the single gene analyses; see Holotype MCZR OLIGOCHAETA 0205, whole-mounted “Results”). For COI, the sequences of specimens from the specimen, fixed in Bouin Feb 2016. Gladkoye site were included (see sub-section on sequencing above), unfortunately H3 was not available for these speci- Type locality Elovoye (56° 8′ 40.08″ N, 92° 32′ 27.97″ E), mens. Xml input files were created in BEAUTI v1.8.2 village near Yemelyanovo International Airport, 25 km NWof (Drummond et al. 2012). The following settings were used: Krasnoyarsk, soil within and around greenhouse (loc. 4). 296 Table 1 List of specimens used in the molecular analyses, with collection data, specimen identification codes, GenBank and Museum accession numbers. In bold are new sequences generated in this study and Museum numbers for paratypes (P). The voucher of H. heleotropha is deposited in ZMBN (University Museum Bergen, Norway)

Species ID Locality Coordinates COI 12S 16S H3a H3b 18S 28S Museum Acc. n. NE

Henlea petushkovi 47 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382709 MH382649 MH382661 - - MH382672 - sp. n. Gladkoye H. petushkovi sp. n. 48 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382712 MH382657 MH382668 - - MH382673 - Gladkoye H. petushkovi sp. n. 49 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382703 MH382655 MH382664 - - MH382674 - Gladkoye H. petushkovi sp. n. 50 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382704 MH382650 MH382666 - - MH382675 - Gladkoye H. petushkovi sp. n. 51 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382705 MH382656 MH382669 - - MH382677 - Gladkoye H. petushkovi sp. n. 52 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382721 MH382653 MH382662 - - MH382678 - Gladkoye H. petushkovi sp. n. 53 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382706 MH382654 MH382665 - - MH382679 - Gladkoye H. petushkovi sp. n. 54 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382707 MH382651 MH382667 - - MH382680 - Gladkoye H. petushkovi sp. n. 55 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382708 MH382652 MH382663 - - MH382681 - Gladkoye H. petushkovi sp. n. 56 Russia: Krasnoyarsk, 56°21'33.27" 92°26'7.51" MH382720 --- - MH382676 - Gladkoye H. petushkovi sp. n. CE28001 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382713 --MH382747 MH382761 - MH382690 MCZR OLIG Elovoye 0218 H. petushkovi CE31467 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382714 --MH382752 MH382746 - MH382698 MCZR sp.n.(P) Elovoye OLIG 0206 H. petushkovi CE31468 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382711 --MH382748 MH382763 - MH382691 MCZR sp.n.(P) Elovoye OLIG 0207 H. petushkovi CE31469 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382715 --MH382749 MH382758 - MH382692 MCZR sp.n.(P) Elovoye OLIG 0208 H. petushkovi CE31470 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382716 --MH382753 MH382764 - MH382699 MCZR sp.n.(P) Elovoye OLIG 0209 H. petushkovi CE31475 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382718 --MH382756 MH382759 - MH382696 SMNH Type sp.n.(P) Elovoye 9076

H. petushkovi CE31476 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382719 --MH382750 MH382765 - MH382701 SMNH Type al. et E. Rota sp.n.(P) Elovoye 9077 H. petushkovi No such Russia: Krasnoyarsk, 55°51'20.8" 92°32'50.69" SMNH Type sp.n.(P) no. Mana river 9078 w e bioluminescent new Two Table 1 (continued)

Species ID Locality Coordinates COI 12S 16S H3a H3b 18S 28S Museum Acc. n. NE

Intermediate spm1 CE31471 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382717 --MH382754 MH382760 - MH382700 MCZR OLIG Bolshie Koty 0219 Intermediate spm2 CE31477 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382727 --MH382742 MH382768 - MH382697 MCZR OLIG Henlea Bolshie Koty 0220

Henlea rodionovae CE27993 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382722 -- 297 MH382745 MH382767 - MH382687 MCZR Siberia from Enchytraeidae) (Clitellata, sp.n.(P) Bolshie Koty OLIG 0215 H. rodionovae CE27994 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382723 --MH382744 MH382757 - MH382686 MCZR sp.n.(P) Bolshie Koty OLIG 0216 H. rodionovae CE27995 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382724 ----- MH382688 MCZR OLIG sp. n. Bolshie Koty 0221 H. rodionovae CE27997 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382735 ------MCZR OLIG sp. n. Elovoye 0222 H. rodionovae CE27998 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382710 ----- MH382689 MCZR OLIG hybrid Elovoye 0223 H. rodionovae CE27999 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382728 --MH382751 - - MH382702 MCZR OLIG sp. n. Elovoye 0224 H. rodionovae CE31472 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382725 --MH382743 MH382762 - MH382693 SMNH Type sp.n.(P) Bolshie Koty 9079 H. rodionovae No such Russia: Irkutsk, 51°54'23.5" 105°4'32.1" SMNH Type sp.n.(P) no. Bolshie Koty 9080 H. rodionovae CE31473 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382726 --MH382755 MH382766 - MH382694 MCZR OLIG sp. n. Bolshie Koty 0225 H. rodionovae CE31474 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382729 ----- MH382695 MCZR OLIG sp. n. Bolshie Koty 0226 Henlea sp. A CE27996 Russia: Irkutsk, 51°54'23.5" 105°4'32.1" MH382734 --MH382736 - - MH382683 MCZR OLIG Bolshie Koty 0227 Henlea sp. B CE28002 Russia: Krasnoyarsk, 56°8'40.08" 92°32'27.97" MH382733 --MH382737 - - MH382684 MCZR OLIG Elovoye 0228 H. glandulifera CE2629 Sweden: Öland, 56°59'34.6" 16°52'35.3" MH382730 MH382658 MH382670 - - MH382682 MH382685 SMNH S. Greda 169924 H. heleotropha CE24429 Norway: 67°15'56.2" 15°17'38.0" MH382732 MH382659 MH382671 MH382741 - - -ZMBN Nordland, Törresvik 111274 H. 658 S. Korea: 35º50'59.0" 127º07'56.4" KR8723201 --KR8723571 - - - magnaampullace- Jeollabuk-do, a Jeonju-si 668 35º46'04.6" 126º43'14.8" KR8723211 --KR8723581 - - - 298 Table 1 (continued)

Species ID Locality Coordinates COI 12S 16S H3a H3b 18S 28S Museum Acc. n. NE

H. S. Korea: magnaampullace- Jeollabuk-do, a Buan-gun H. montana CE21004 Norway: 60°16'30.0" 5º20'3.5" MH382731 MH382660 - MH382739 - - -SMNH Hordaland, Fana 169925 H. nasuta CE824 Sweden: Västergötland, 58°37'13.6" 13°25'59.7" GU9020832 GU9020832 GU9018232 KX6448833 - GU9019082 GU9019942 Hällekis H. ochracea 249 USA: Alaska, Fairbanks --KR8723291 --KR8723611 - - - H. ochracea 253 USA: Alaska, Fairbanks --KR8723301 --KR8723621 - - - H. perpusilla CE853 Sweden: Västergötland, 58°34'59.1" 13°25'96.5" GU9020842 GU9017332 GU9018242 MH382738 - GU9019092 GU9019952 Österplana H. ventriculosa CE1021 Sweden: Bohuslän, 58°26'0.0" 11°34'8.0" GU9020842 GU9017342 GU9018252 KU8942814 - GU9019102 GU9019962 Ingalsröd H. groenlandica 381 Norway: Svalbard, --KR8723251 --KR8723601 - - - Russelbukta H. groenlandica 432 Norway: Svalbard, --KR8723241 --KR8723591 - - - Kapp Haiglin Bryodrilus CE19814 Norway: Hedmark, 61°39'24.84" 11°48'59.04" KX6187353 KX6187453 KX6187593 - - KX6187733 KX6187913 diverticulatus Engerdal Claparedrilus CE23750 Norway: Nordland, 68°13'29.28" 16°04'37.56" KU8940974 KU8627204 KU8627224 KU8942154 - KU8627884 KU8628224 semifuscoides Bognes Globulidrilus CE2922 Sweden: Öland, 56°51'43.6" 16°51'14.1" KX6187323 KX6187433 KX6187623 MH382740 - KX6187703 KX6188003 riparius Melösavik Marionina CE811 Sweden: Västergötland, 58°24'8.64" 13°38'27.96" GU9020982 GU9017482 GU9018392 KU8942864 - GU9019232 GU9020112 communis Varnhem Oconnorella CE788 Sweden: Västergötland, 57°59'68.3" 12°35'96.7" GU9021052 GU9017572 GU9018482 KX6448853 - GU901932 GU9020212 cambrensis Vårgårda

1 From Dózsa-Farkas et al. (2015); 2 From Erséus et al. (2010); 3 From Martinsson et al. (2017); 4 From Klinth et al. (2017a) oaE tal. et E. Rota Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 299

Fig. 3 Henlea petushkovi sp. n. Different views of prostomium in living worms. a dorsal. b lateral. c dorsolateral. d ventrolateral. Scale bars: a–d 200 μm

Paratypes MCZR OLIGOCHAETA 0206–0209, four posteri- Etymology Named for Valentin N. Petushkov, researcher of orly amputated submature or immature specimens (vouchers the Russian Academy of Sciences, for his lifelong dedication CE31467–CE31470) preserved in 85% ethanol from type lo- and excellent accomplishments in the biophysical study of cality (loc. 4), Apr 2017; posterior parts used for DNA extrac- enchytraeid bioluminescence. tion (COI Barcodes, Nuclear markers and GenBank nos. listed in Table 1). MCZR OLIGOCHAETA 0210, mature specimen External Colour white-yellowish, emitting flashes of bluish from forest soil by Kacha river (loc. 1), fixed in Bouin Oct 2001, light upon stimulation. Body wall opaque, rather stiff. Live anterior 16 segments cross-sectioned, posterior body (49 seg- body size: length 25–30 mm, width at V 720 μm, at clitellum ments) in ethanol. MCZR OLIGOCHAETA 0211, submature 750–850 μm. Fixed dimensions can be much altered by body specimen from same locality (loc. 1), fixed in Bouin Oct 2001, contraction: length 16–21 mm; width at V 800–1200 μm, at anterior 20 segments sectioned longitudinally, posterior body clitellum 900–1500 μm. Segments 61–70, average 65.5 (n = (45 segments) in ethanol. MCZR OLIGOCHAETA 0212– 8). Prostomium hemispherical, in a side view dorsally de- 0213, two mature specimens, whole-mounted, from forest soil pressed, short, in vivo 240 μmfromheadporetotip, along Mana river (loc. 2), fixed in 80% ethanol Aug 2003. 400 μmwideatbase(Fig.3a, b, c, d). Peristomium (segment SMNH Type Collection 9076, one mature specimen, whole- I) in vivo 250–280 μm long, 550 μmwide(Fig.3a, b, c, d); mounted, from forest soil along Mana river (loc. 2), fixed in after fixation 190–200 μm long, 530–650 μm wide. Head 80% ethanol Aug 2003. SMNH Type Collection 9077–9078, pore as a transversal slit at 0/1. Epidermal gland cells irregu- two posteriorly amputated submature or immature specimens larly shaped and sized, distributed in 4 transversal rows. (vouchers CE31475–CE31476) preserved in 85% ethanol from Chaetae arranged in lateral and ventral bundles, straight, type locality (loc. 4), Apr 2017. (1)2÷5 − 2÷5(6,7,8) : (1)2÷6 – 2÷7(8) per bundle, most nu- merous in segments XL–L (Suppl. Fig. 1); the outer chaetae in Additional material Ten specimens (vouchers not preserved) a bundle larger than the inner ones (e.g., 115 μmvs90μmin from farmyard soil at Gladkoye (loc. 3), DNA-sequenced Mar segment XVII), length up to 150 μm (caudally), thickness up 2015 (COI Barcodes, other markers and GenBank nos. listed to 13 μm. Chaetae of XII missing in mature specimens. in Table 1). Numerous specimens from loc. 1, 2, and 4 in first Clitellum (Fig. 4g, h) over 1/4 XI–3/4 XIII, moderately ele- author’s collection. vated, made of small gland cells, the hyaline cells somewhat 300 Rota E. et al.

Fig. 4 Henlea petushkovi sp. n. Internal anatomy shown by histological sections. a Longitudinal oblique section of segments V–IX (arrows point to the branches of oesophageal appendages in VI and VII). b Longitudinal parasagittal section through the gut diverticula in VIII, showing the anterior connection to septum 7/8. c–e Longitudinal sections through a spermatheca, showing: c the glands (gl) surrounding the distal end of ectal duct; d the bell- shaped widening of the ectal duct canal at the junction with ampulla; e the ampulla and ental duct joining gut close to septum 5/ 6. f Sperm funnel. g, h Arrangement of clitellar glands, from tangential sections of XII (g dorsal) and XI–XIII (h ventral). Scale bars: a, b, h 200 μm; c–g 100 μm

larger than the granular ones, the two types irregularly ar- length 360–410 μm), and the frontal border appears doubly ranged. Paired male pores ventral in XII, inside deep male paired, with diverging anterior tips (middorsally and bursae; clitellar gland cells absent between them. No copula- midlaterally the anterior outline appears concave; Fig. 5a, b, tory glands. Paired spermathecal pores in lateral lines at 4/5. c). Chloragogen cells from IV, small, forming a dense olive- browntissuefromX;invivogranules irregularly distributed Internal Brain concave anteriorly, deeply indented posteriorly, and cells appearing more aggregated in places, so as to make slightly longer than wide, ca. 170 μmwideand180μmlong the tissue not uniformly opaque. Dorsal vessel originating in IX (Fig. 3a, d). Septa 6/7–8/9 thickened (Figs. 4a, b, and 5b). (Figs. 4a, and 5a), with heart-like expansions in IX, VIII and Oesophageal appendages adhering to gut wall and, through VII; lateral commissures seen in IV (2 pairs) and V (1 pair) (Fig. VI and beginning of VII, forming frilled ridges giving off nu- 4a, d, e, arrowheads); cell nuclei seen on the inner wall of all merous branched extensions into the coelom dorsally and ven- vessels. Preclitellar nephridia 6 pairs, in 5/6–10/11, efferent trally (Fig. 4a, arrows). Intestinal diverticula forming a ducts arising anteroventrally (Fig. 4a). Coelomocytes abundant, multitubular tulip-shaped structure surrounding gut and occu- with evenly fine-granulated cytoplasm, opaque when accumu- pying much of the coelomic cavity in VIII (Figs. 4a, and 5a–c); lated (Fig. 5b, c), discoid, 60–85 μmacrossbothinvivoand tubules arising from intestine in posterior of VIII, extending fixed (and regardless of worm’s maturity), longer than half the forwards while branching, ending blindly to make the frontal largest outer chaetae (Fig. 5d); live coelomocytes often showing border of the organ (Fig. 4a, b). The mass of tubules adheres to a peripheral annular wrinkle (Fig. 5d), but no cell sculpture gut but anteriorly is extensively anchored to septum 7/8 remains evident in whole-mounts or sections (Fig. 4a, d, f). (Fig. 4b), thus, the diverticula appear evenly expanded along Seminal vesicles paired, extending to X (Fig. 4f). Sperm anterior half (in vivo maximum diameter ca. 490–520 μm, funnels cylindrical, slightly narrowing entally to ectally, in vivo Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 301

Fig. 5 Henlea petushkovi sp. n. Internal anatomy as seen in living worms. a–c Different views of gut diverticula in VIII (a dorsolateral. b dorsal. c ventrolateral); white arrows indicate the thickened septum 7/8. d Close-up of nephridium and coelomocytes. Scale bars: a–d 200 μm

ca. 400 μm long and 120 μm wide (fixed: 375 by 150 μm); indented, dorsal blood vessel from IX, conspicuous collar low, wider than ental end of funnel body (Fig. 4f), often spermathecal ectal glands and nephridia from 5/6. As con- with upturned border. Heads (i.e., nuclei) of spermatozoa about cerns the number of chaetae and body segments, the new 48 μm long. Vasa deferentia confined to XII, long, forming species is most similar to H. tubulifera Welch 1914b, original- regular loops, relatively thin (in vivo ca. 12 μm thick) (Fig. ly described from North America (Michigan). However, in 4f). Penial bulbs simple, of lumbricilline-type (i.e., with a com- Welch’s species the coelomocytes were smaller (36–54 μm), pact glandular body surrounded by muscles). Spermathecae the oeosophageal appendages ended posteriorly in VI and one pair, in V. Three or four large accessory gland cells around had only dorsal extensions into the coelom, the sperm each spermathecal pore; glands elongate, club-shaped, attached funnels showed different size and shape, and so did the by their narrower ends to ectal pore, 100–140 μm long and spermathecae. On the other hand, H. moderata Welch 40 μminmaxwidth(Fig.4c). Ectal ducts stout, in vivo 300– 1914a from Illinois resembles the new species in the size 350 μm long and 60 μm wide, thick-walled (ca. 20 μm), with of coelomocytes (max length 85 μm), and the structure of inner canal expanding markedly (from 10 to ca. 35 μm) from the oeosophageal appendages (giving off branches in VII), pore to spermathecal midcourse; at junction with ampulla, duct but differs from the new species in its lower segment canal forms a bell-shaped cavity 50 μm across, merging with number, shorter sperm funnels, and differently shaped ampullar lumen (Fig. 4d). Ampullae oval, 140 μmwide,not brain and spermathecae (see Welch 1914a). Neither of clearly demarcated from sac-like ental ducts, total length of these named species was reported by Welch to be ampulla and ental duct 250–300 μm, entally merging into a bioluminescent. short common chamber communicating with oesophagus in As shown and discussed below, H. petushkovi sp. n. is most posteriorofV(Fig.4a, e). Spermatozoa present all along the closely related to H. rodionovae sp. n. described below, and ectal ducts, in the lumen and walls of ampulla and in ental ducts there is genetic evidence that the two species are capable of (Fig. 4a, d, e). hybridizing.

Taxonomic relationships Henlea petushkovi sp. n. belongs to Henlea rodionovae sp. n. (Figs. 6–7) a non-monophyletic subgroup of Henlea discussed below, “Hepatogaster” (Suppl. Table 1), characterized by a Holotype MCZR OLIGOCHAETA 0214, whole-mounted, multitubular gut outgrowing in VIII, brain posteriorly mature specimen, fixed in Bouin Nov 2015. 302 Rota E. et al.

Fig. 6 Henlea rodionovae sp. n. Morphology of prostomium and body wall in living worms. a–d, f, g Different views of prostomium (a, g dorsal. b, c ventral. d, f ventrolateral). e Body wall of segments III–V. Arrows point to rows of epidermal glands. Scale bars: a–g 200 μm

Type locality Bolshie Koty (51° 54 ′23.5″ N, 105° 04′ 32.1″ ethanol, Apr 2017; posterior parts used for DNA extraction E), a former gold-mining site now belonging to Pribaikalsky (COI Barcode, Nuclear markers and GenBank nos. listed in National Park, 70 km from Irkutsk, soil inside and immediate- Table 1). ly outside greenhouse (loc. 6). Additional material One whole-mounted, submature specimen Paratypes All from the type locality (loc. 6). MCZR from forest soil by Kacha river (loc. 1), fixed in Bouin Jan OLIGOCHAETA 0215–0216, two posteriorly amputated 2002. Material not preserved from Listvyanka (51° 50′ 52.7″ specimens (vouchers CE27993, CE27994) preserved in 85% N, 104° 52′ 43.0″ E), near the southern tip of Lake Bajkal, soil ethanol, Nov 2015; posterior parts used for DNA extraction within and around greenhouse (loc. 5), Aug 2015. Numerous (COI Barcodes, Nuclear markers and GenBank nos. listed in specimens from loc. 6 in first author’s collection. Table 1). MCZR OLIGOCHAETA 0217, one mature speci- men, whole-mounted, unstained, Dec 2015. SMNH Type Etymology Named for Natalja S. Rodionova, researcher of the Collection 9079, one mature specimen, whole-mounted, Russian Academy of Sciences, for her lifelong dedication and Nov 2015. SMNH Type Collection 9080, one posteriorly excellent accomplishments in the biochemical study of amputated specimen (voucher CE31472) preserved in 85% enchytraeid bioluminescence. Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 303

Fig. 7 Henlea rodionovae sp. n. Internal anatomy as seen in living worms. a, b Dorsal view of segments VI–X and close-up of gut diverticula in VIII. c, d Ventral view of segments VI–X and close-up of gut diverticula in VIII. e, f Dorsal and ventral views of oesophageal appendages and their branches (arrows) in VI. g Close-up of nephridium (black arrow, nephrostome; white arrow, efferent duct) and coelomocytes (cc). h Spermatheca. j Sperm funnel. Scale bars: a–j 200 μm

External Colour yellow with a shade of orange-brown, emitting Head pore as a transversal slit at 0/1. Epidermal gland cells flashes of bluish light upon stimulation. Body wall relatively small, irregularly shaped, forming 3 complete rows (Fig. 6d, soft, transparent, contractile. Live body size: length 20–24 mm, e, f, arrows). Chaetae straight, arranged in lateral and ventral width at V 650–680 μm, at clitellum 750–850 μm, posterior bundles, 2÷4(5) – 2÷7 : 2÷4(6) – 2÷7, most numerous around half of body distinctly tapering caudad. Fixed dimensions: segments XL; the outer chaetae in a bundle larger than the length 14 mm; width at V 650–720 μm, at clitellum 730– inner ones (Suppl. Fig. 2), length up to 130–150 μm(caudal- 1000 μm. Segments 60–67, average 64.6 (n = 8). Prostomium ly), thickness up to 12 μm. Chaetae of XII missing in mature blunt conical, narrow at base, as long as wide, in vivo 200– specimens. Clitellum over 1/4 XI–3/4 XIII, slightly elevated. 230 μm from head pore to tip, very mobile (Fig. 6a, b, c, d, f, Paired male pores ventral in XII, inside deep male bursae; g), on all sides incised by grooves, some of which transversal, clitellar gland cells absent between them. No copulatory longitudinal or radial; grooves vary in position between speci- glands. Paired spermathecal pores in lateral lines at 4/5. mens; longitudinal grooves extend ventrally also all along peristomium (Fig. 6b, f); grooves visible also after fixation. Internal Brain (Fig. 6g) concave anteriorly, deeply indented Peristomium (segment I) in vivo 250–280 μm long, 380– posteriorly, longer than wide, ca. 140–160 μmwideand190– 520 μm wide; after fixation 130–150 μm long, 450 μmwide. 210 μm long. Septa 6/7–8/9 thickened. Oesophageal 304 Rota E. et al. appendages adhering to gut wall, forming frilled ridges, giving Arctic. However, the latter species was originally described with off numerous (5–6 per side) branched extensions into coelom a smaller size (15 mm, 52 segments) and with efferent duct dorsally and ventrally in VI (Fig. 7c, e, f, arrows). Intestinal arising from the posterior end of the nephridial body. Later, diverticula as a multitubular apple-shaped structure surround- Welch (1919) gave a more extended description of Eisen’s taxon ing gut and occupying much of the coelomic cavity in VIII, based on material from the Northwest Territories of Canada, a length 300–320 μm, maximum cross diameter at midlength, description that confirmed the aberrant nephridial morphology. ca. 400–500 μm(Fig.7a, b, c, d); mass of tubules symmetri- Despite these morphological similarities with species known cally paired, arising from intestine near 8/9, extending for- from other parts of the northern hemisphere, this study shows wards adhering to gut, midlaterally the anterior border is con- that H. rodionovae sp. n. is most closely related to H. petushkovi cave. The tubules end blindly against cephalic border of the sp. n., and that these two taxa can even hybridize with each other. organ. Chloragogen cells from IV, finely granular, forming a dense opaque orange-brown or chestnut-brown tissue from X Morphologically intermediate specimens (Fig. 7a, c). Dorsal vessel originating in IX (Fig. 7b), with heart-like expansions in IX, VIII and VII. Preclitellar Two of the sequenced luminous specimens from Bolshie Koty nephridia 6 pairs, in 5/6–10/11, efferent duct arising greenhouse showed intermediate morphological patterns be- anteroventrally (Fig. 7g). Coelomocytes abundant, with dis- tween the above described new species. Specimen CE31471 tinct nucleus and evenly fine-granulated cytoplasm, discoid, had the tulip-like gut diverticula characterizing H. petushkovi shorter than half the largest outer chaetae (Fig. 7d, g), in vivo sp. n., combined with the small coelomocytes (40–60 μm long 40–50 μm, at most 60 μm across; after fixation, 30–35 μm. in vivo) and furrowed prostomium typical of H. rodionovae Seminal vesicles X–XI. Sperm funnels elongate conical, sp. n. On the other hand, specimen CE31477 possessed apple- narrowing entally to ectally, in vivo ca. 400 μm long and like diverticula as in H. rodionovae sp. n., and large 110 μm wide; collar 20 μm high, wider than the body of coelomocytes (up to 75 μm long in vivo) and rounded prosto- funnel, reflected (Fig. 7j). Heads (i.e., nuclei) of spermatozoa mium as in H. petushkovi sp.n. about 50 μm long. Vasa deferentia confined to XII, long, forming many regularly loops, relatively thin (in vivo 13– Unidentified Henlea species 15 μm thick) (Fig. 7j). Penial bulbs simple, small, of lumbricilline-type. Spermathecae one pair, in V (Fig. 7h). A few non-bioluminescent specimens of Henlea (including Some accessory gland cells at each spermathecal pore; glands one sequenced from Bolshie Koty and one sequenced from elongate, club-shaped, attached by their narrower ends to ectal Elovoye greenhouses) belong probably to two different un- pore, each gland 75 μm long, 40 μm wide at bottom. Ectal known species. Because of the low number of preserved spec- ducts stout, cylindrical, in vivo 200–300 μmlongand60μm imens and inadequate maturity, we leave them unnamed, wide at pore, thick-walled (ca. 15 μm), with inner canal waiting for additional material, but provide their characteriza- expanding markedly from pore to spermathecal midcourse tion (and for sp. A, a thorough illustration), to allow a com- (from 6 to ca. 30 μm). Ampullae oval, in vivo 100 μm wide, parison with the coexisting taxa. In the following short de- externally not clearly demarcated from ectal ducts. Walls of scriptions, important features are emphasized in boldface. ampullae ca 15 μm thick. Ental ducts 280 μm long, spindle sac-like, merging into a common chamber communicating Henlea sp. A (Fig. 8) Three specimens (2 mature and 1 with oesophagus in posterior of V (Fig. 7h). submature) examined alive from Bolshie Koty greenhouse, of which one (mature) preserved and sequenced (CE27996). Taxonomic relationships Henlea rodionovae sp. n. belongs to Non luminous. Large worms, 26 by 0.87 mm, with 66–76 thesamegroupofHenlea as H. petushkovi sp.n.,i.e.,itpossesses segments (Fig. 8a). Lateral and ventral chaetae up to 6 Hepatogaster-like gut diverticula in VIII consisting of masses of preclitellarly, up to 8 postclitellarly (Fig. 8h), maximally branched tubules (see Suppl. Table 1). Morphology and DNA 150 μm long. Prostomium with dorsal sinuous transverse (see below) indicate that the two species are very closely related, furrow (epilobic closed) (Fig. 8a, b, c). Brain trapezoidal, but differ from each other by the body wall strength, the structure incised posteriorly (Fig. 8c). Oesophageal appendages as of prostomium, brain shape, size of coelomocytes and structure frilled ridges forming unpaired (middorsal and midventral) of intestinal diverticula. The number of chaetae and body thick, ear-like loops in anterior of V (Fig. 8a, d) and free segments as well as the size of coelomocytes makes H. branches in VI (Fig. 8e). Gut diverticula Hepatogaster-like, rodionovae most similar to H. tubulifera from Michigan, but consisting of four large multitubular outgrowings fused to- the oeosophageal appendages, the sperm funnels and the gether and surrounding the gut all along segment VIII spermathecae have different structure. The body colour and (Fig. 8a, g). Dorsal vessel from IX. Coelomocytes small, geographical location would instead suggest kinship to H. 35–40 μmlongin vivo (Fig. 8e, f). Nephridia from 5/6. ochracea, described by Eisen (1878, 1879) from the Siberian Large seminal vesicles, occupying XI–XII (Fig. 8a). Sperm Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 305 funnels 350 by 100 μm, with wide collar; penial bulbs large, luminous, whitish. Large (25 mm long; 69 segments). 260 μmlong(Fig.8a). Spermathecal ampulla conical. – This Body wall soft. Chaetae up to 6 per bundle, maximally species shows a unique combination of characters (in bold 150 μm long. Prostomium smooth. Brain 200 μm long in face above). The midgut structure, dorsal vessel origin and vivo, 175 by 90 μm after fixation, posteriorly indented. Gut the features of the reproductive organs are similar to those of diverticula leptodera-like,i.e.,in the form of two large, H. petushkovi sp. n., but the oesophageal appendages show a fan-shaped pouches projecting freely forward from 8/9 condition so far known only in H. nasuta (=Enchytraeus through segment VIII. Dorsal vessel from VIII. leptodera Vejdovský, 1879). The coelomocytes appear excep- Coelomocytes finely granular, opaque, more than 50 μm long tionally small and the morphology of the prostomium is in vivo. Nephridia from 5/6. Seminal vesicles absent. Sperm unique in the genus. funnels short, conical Spermathecae not fully developed. – This immature specimen resembles in many somatic aspects H. Henlea sp. B One specimen (submature) preserved and se- nasuta sensu lato, but differs by the thinness of the body wall and quenced (CE28002) from Elovoye greenhouse. Non the more elongate brain (see e.g., Rota 1995).

Fig. 8 Henlea sp. “A”,living worm. a Ventral view of segments I–XIV (white arrow, oesophageal appendage). b, c Prostomium, showing its peculiar sinuous furrow. d, e Close-up of oesophageal appendages forming a thick middorsal loop in anterior of V (d within circle) and giving off branches in VI (e). f Coelomocytes. g Gut diverticula in VIII. h Ventral chaetal bundle in a postclitellar segment. Scale bars: a 500 μm, b–h 200 μm 306 Rota E. et al.

Molecular results only one of the 14 specimens of the new species for which H3 was successfully sequenced (Table 1), only one of the two Genetic analyses copies was recovered. In the whole alignment, including both paralogues, seven bases were variable; in H3a five bases were COI was successfully sequenced from all 20 Siberian speci- variable, and in H3b two bases were variable. In 28S, only one mens, H3 from 16 specimens, and 28S from 19 specimens base out of 306 was variable in one of the two new species (Table 1). After trimming, the COI alignment was 658 bp long (one autapomorphy in a single specimen). for the two new species, with the ten specimens from Gladkoye included (see “Material and methods”), and 84 ba- Phylogenetic estimation ses were variable. The H3 alignment was 312 bp long. In the two new species two H3 paralogues are present, the gene In both the COI and H3 analyses, the effective sample size duplication is recent and all substitutions are synonymous, (ESS) was large (> 200) for all parameters. In the COI tree i.e., the sequences still code for the same amino acids. In the (Fig. 9a) two main clades are found, both with maximum results below we refer to the paralogues as H3a and H3b. In support. These clades largely correspond with the two new

Fig. 9 Single gene trees and haplotype networks. a, b Gene-trees (a COI, haplotype networks (c COI, d H3). The size of the circles represents the b H3) estimated with Bayesian coalescent analysis in BEAST. Numbers frequency of the haplotype among the sampled specimens, colours above branches are posterior probabilities. Scales show expected indicate the different species as shown in the legend, and the hatch numbers of substitution per site. The specimen shaded in red has a marks correspond to substitutions between haplotypes conflict between morphology and COI data. c, d Statistical parsimony Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 307 species, but one H. rodionovae-looking specimen (CE27998) paralogue, H3b, the sequences from the two species are is found in the clade otherwise containing only H. petushkovi mixed. In the H3 tree, the support values are lower than in worms. Furthermore, the two morphologically intermediate the COI tree, reflecting its much shorter branches (fewer nu- specimens (CE31471 and CE31477) are found in one clade cleotide substitutions in the dataset). each. In the H3 tree (Fig. 9b) there is a basal split separating In the concatenated tree based on six genes (Fig. 10), Henlea the two paralogues. H3a, the more variable of the two, is is not recovered as monophyletic, as one of the outgroups, further divided into two groups which could correspond with Oconnorella cambrensis, is found in a clade together with the two species, but one H. petushkovi specimen (CE28001) is Henlea magnaampullacea and H. sp. B, but with low support found in the mostly H. rodionovae clade, and two H. (PP = 0.84). There is no support for the monophyly of rodionovae specimens (CE31473 and CE27999) are found Hepatogaster in the tree. Of the five species morphologically in the mostly H. petushkovi clade. Furthermore, the two mor- corresponding with Hepatogaster (Henlea ochracea, H. phologically intermediate specimens are found one in each magnaampullacea, H. petushkovi, H. rodionovae,andH.sp. clade (CE31471 in the mostly H. petushkovi clade; and A), only Henlea petushkovi and H. rodionovae are sister species CE31477 in the mostly rodionovae clade). In the other (PP = 1) and together found as sister to H. sp. A, although

Fig. 10 Phylogeny of Henlea based on combined COI, H3, 12S, 16S, conflict between morphology and COI data, and it is named based on 18S, and 28S data from 13 Henlea species and five outgroups estimated morphology. Numbers at branches are posterior probabilities. Scale using Bayesian Inference in MrBayes. The specimen shaded in red has a indicates expected numbers of mutations per site 308 Rota E. et al. without support (PP = 0.64). Henlea ochracea is found in a clade are found, but the separation is not complete. In the less together with H. groenlandica and H. ventriculosa (PP = 1), and variable paralogue, H3b, however, there is no such separa- as mentioned above, H. magnaampullacea is found together tion between the two species. The congruence between mor- with Oconnorella cambrensis and H. sp. B. No other relation- phology and COI data suggests that the two groups are sep- ships within Henlea are supported. arately evolving lineages. The less complete separation in H3a and lack of separation in H3b could be explained by a Haplotype networks combination of lower substitution rate and larger effective population size in the H3, compared to the mitochondrial The haplotype networks show the same general patterns as the COI, resulting in incomplete lineage sorting between the gene trees. In the COI network (Fig. 9c), the two species are two species. The species could be considered as being in separated, with the exception of CE27998, which is found the “gray zone” (cf. de Queiroz 2007,fig.1),wheredifferent among the H. petushkovi haplotypes despite morphologically data and different criteria can yield different species bound- being an H. rodionovae, and one morphologically intermediate aries. However, the combination of COI and morphology is, specimen is found within each species. In the H. petushkovi according to us, enough to delimit H. rodionovae sp. n. and group, there are 10 haplotypes, one unique for CE27998 men- H. petushkovi sp. n. as two separate species. tioned above, and four in the H. rodionovae group. The conflict between morphology and COI data The H3 network (Fig. 9d) consists of two main groups, (CE27998) suggests that H. rodionovae sp. n. and H. separating the two paralogues. For H3a, the haplotypes petushkovi sp. n. are able to hybridize, and that they are not are to some extent sorted according to the morphology, completely reproductively isolated. Interestingly, according to with the exception of three specimens—as in the H3 tree. Christensen (1961) H. jutlandica Nielsen and Christensen, The two intermediate specimens (CE31471 and CE31477) 1959, originally regarded as a tetraploid cytotype n =34of are found in the same groups as in the COI and H3 trees. H.ventriculosa, is an amphimictic alloploid with normal mei- For H3b, there are three haplotypes, two of them shared osis, arisen through hybridization. There is also at least one by H. rodionovae and H. petushkovi, and one of these two additional case of hybridization between enchytraeids, in the is present in the intermediate specimens. genus Hemifridericia Nielsen and Christensen, 1959, based on DNA evidence (Martinsson and Erséus 2018). Genetic distances Hybridization cases are also documented between several spe- cies in the earthworm family Lumbricidae (Dupont et al. In COI, the uncorrected p-distances within the H. petushkovi 2016; Martinsson and Erséus 2017; Plytycz et al. 2018). clade vary from 0.0 to 1.2%, and within the H. rodionovae Henlea rodionovae and H. petushkovi are likely sister spe- clade between 0.0 and 2.3%, and between the two species cies that have emerged in allopatry, but which seem not to from 10.2 to 11.6%. These distances exclude specimen have evolved complete reproductive isolation. After coming CE27998, which is morphologically a H. rodionovae,while into secondary contact again the lineages may have started to its COI corresponds to that of H. petushkovi. hybridize. The third genetic marker, 28S, does not have enough variationtobeusedforseparatingthetwospecies;thepart Discussion of 28S used in this study is evolving too slowly. 28S se- quences are known to be identical between closely related Species delimitation species, e.g., in some species pairs of the enchytraeid genera Lumbricillus Ørsted, 1844 and Chamaedrilus Friend, 1913 The cases of conflict between morphology and the COI and (data from Klinth et al. 2017a;Martinssonetal.2017). H3 sequences raise the question, whether H. rodionovae The Gladkoye population, which is from a wooded farm sp.n.andH. petushkovi sp. n. should be treated as separate yard, seems to be more diverse than the other populations species or not. Using the unified species concept by de found in and around greenhouses. This could be due to a Queiroz (2007), we define species as separately evolving reduction in genetic diversity caused by a genetic bottleneck metapopulation lineages, and assess their separation using when relatively few specimens founded the greenhouse pop- both morphological and genetic data. The two groups are ulations. More data are needed to test this. separated morphologically, with the exception of a few in- termediate specimens. Further, they are separate in COI, but Autochthony of bioluminescence one specimen morphologically identified as H. rodionovae is genetically clustered together with the specimens of H. Considering the old records of Henlea-type bioluminescence petushkovi. In the more variable of the two H3 paralogues, in European greenhouses (see “Introduction”), and the in- H3a, two clades largely corresponding with the two species volvement of greenhouse populations in Siberia, the question Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 309 arises of the potential geographic source of the studied spe- low support in our tree. The low gene coverage may also be cies. The first luminous Siberian Henlea worms investigated the explanation for not recovering Henlea as monophyletic. biophysically and morphologically (and belonging to both Further, for our phylogeny we only sampled 13 of the over 30 new species, with a dominance of H. petushkovi)werecol- accepted species of Henlea (Schmelz and Collado 2012). lected in wild forest soil along Kacha and Mana rivers (habitat as described in Rota et al. 2003), and some worms sequenced Taxonomy of luminescence in Moscow and clustering with petushkovi (in the COI tree) came from a wooded countryside context (Gladkoye). In all Among the species regarded as valid in Henlea, none has been these localities, the Henlea worms co-inhabited the soil with reported to have a grooved prostomium (which characterizes, Fridericia heliota, a species endemic to the Siberian taiga. in different manners, H. rodionovae sp. n. and species A), and Other petushkovi specimens and the majority of rodionovae no member of the Hepatogaster subgroup has ever been re- worms included in this study came from two greenhouses, one ported as capable of bioluminescence (which characterizes H. in the Krasnoyarsk and the other in the Irkutsk region. These rodionovae sp. n. and H. petushkovi sp.n.).Previousrecords greenhouses have a domestic horticultural use and the culti- of the phenomenon were assigned to the species H. vated soil appears to be of local origin. These facts, along with ventriculosa (by Michaelsen in Walter 1909). With regard to the close genetic relationship between the new Henlea spe- this fact, we can be certain about the genus identification, cies, point to them as being autochthonous elements of Michaelsen himself having established Henlea in 1889, but Siberia. Furthermore, the “wild” worms of H. petushkovi were we are doubtful of the species identification, because many morphologically and genetically more consistent than the other species were to be differentiated later in the genus, in- greenhouse specimens. The vegetable trade could be an ex- cluded those ascribable to Hepatogaster, and Michaelsen planation for the mixed, partly hybridized populations. probably identified ventriculosa by exclusion. When Walter Interestingly, F. heliota has never been found in greenhouse (1909) made his observations, Henlea comprised five valid soil (V. N. Petushkov and N. S. Rodionova, in litteris). and four doubtful species (Michaelsen 1900), and H. ventriculosa was the only species with one pair of Status of Hepatogaster Cejka, 1910 spermathecae known to have the dorsal vessel from segment IX and the intestinal diverticula in VIII adhering together and We compared the genetic sequences of our two new Siberian surrounding the gut. No species with Hepatogaster-like gut species and the unnamed species A, all sharing the diverticula had yet been described (see Suppl. Table 1). Hepatogaster-like condition of gut diverticula, with those of Indeed, during our studies, we found no evidence (not even H. magnampullacea recently described from Korea and stated in Siberian specimens) of H. ventriculosa being capable of to possess a “multitubular substructure” (Dózsa-Farkas et al. bioluminescence. Friend (1919)referredtoH. nasuta as being 2015) and also with sequences of H. ochracea provided by the luminous, a citation for which it is impossible to find the same authors, and we found no close affinity. Admittedly, the primary source, and it is not clear whether this was a lapsus position of H. magnampullacea is not clear, as the authors or an emended identification of Walter’smaterial. likened the substructure of its diverticula to that “characteriz- ing” H. ventriculosa, H. jutlandica,andH. groenlandica.In Taxonomic notes on H. nasuta any case, by considering also the other Henlea species includ- ed in our analysis, the molecular phylogeny seems to support a We mentioned above (see “Introduction”), that H. nasuta as transition from smoother diverticula walls via more folded to originally described is different from the definition given by the tubuliferous condition of Hepatogaster as previously sug- Nielsen and Christensen (1959) and based on E. leptodera gested, instead of a sister-group relationship between Vejdovský. Archienchytraeus nasutus Eisen, 1878 was de- Hepatogaster and other Henlea species. Actually, scribed from material collected along river Yenisej, Siberia, Hepatogaster would seem to be an artificial grouping of taxa from 60° 50′ to 72° 40′ N. Eisen (1879) remarked on the large showing similar modifications by convergence (polyphyletic body size (25 by 2 mm, 56 segments), the chaetal bundles grade). The question whether the tubuliferous condition is a containing up to 7 chaetae of unequal size, the large seminal plesiomorphy (as suggested by Tynen et al. 1991,basedon vesicles occupying two segments, and provided illustrations “very preliminary outgroup comparisons”) or a derived con- of the pointed (nasutus) prostomium, the crown of large dition with respect to smoother diverticula needs more data to glands at the ectal opening of the spermathecae and the be resolved with full support. According to our estimated phy- branched oesophageal appendages (“peculiar glandlike or- logeny the tubuliferous condition is apomorphic, and has gans”) near the spermathecal ental end (see Eisen 1879,figs. evolved more than once, but our phylogeny is just a start: as 10 and 66), but did not mention any differentiation of the we tried to maximize the number of species included, we have intestine, nor the place of origin of the dorsal blood vessel. limited gene coverage of some. This probably explains the When Michaelsen (1889) established the genus Henlea,he 310 Rota E. et al. reexamined Eisen’stypesofA. nasutus and compared them Helsinki), they concluded that it comprised a mixture of these with specimens of the coeval European taxon E. leptodera two taxa (Christensen and Dózsa-Farkas 2006). Vejdovský, 1879 (p. 33). He noted several similarities, but opted for keeping the two species separate, on account of the Taxonomic notes on H. irkutensis different internal structure observed in the free intestinal pouches of A. nasutus, comprised of masses of branched tu- In 1929 V. Burov published in Russian (with German summa- bules (Fig. 2c), in comparison to the insignificant wrinkling of ry) an extensive account of a new enchytraeid species, Henlea the wall and the predominant central cavity seen in those of E. (Hepatogaster) irkutensis, found in great numbers in river leptodera (Fig. 2a). He also noted the distinctly unequal chae- woodlands around Irkutsk, eastern Siberia. This species was tal size within bundles of A. nasutus, arranged so that the inner unusual in its quite large size, 24–55 mm length in a fixed chaetae were progressively shorter. In 1900 Michaelsen, how- state, with 60–91 segments, and its thick (up to 100 μmatthe ever, accepted these differences as part of intraspecific varia- clitellum), opaque and midventrally papillate epidermis. The tion and, totally overlooking a further important difference: anatomy reminded in many aspects that of two species de- the conspicuous glands surrounding the spermathecal ectal scribed by Čejka (1910) from the New Siberian Islands, in pores, decided for the synonymization of A. nasutus with E. the Russian Arctic, Hepatogaster birulae and Hg. sibiricus leptodera. Nielsen and Christensen (1959: p. 61) adhered to (see Suppl. Table 1). The latter had been collected in pools this decision but acknowledged: “It can be doubted that the and moist mosses. Henlea irkutensis also came from a semi- species recorded under this name from most European coun- aquatic habitat and therefore was included among the aquatic tries is identical with the Siberian material underlying the Oligochaeta of the USSR treated by Cekanovskaja (1962). It original description”. In the light of the anatomical diversity was transferred to Punahenlea (=red-blooded Henlea)by documented in Henlea during the present study, we believe Nurminen (1980) because of its possession of well- that the identity of A. nasutus should be reconsidered, because developed seminal vesicles. Nurminen already in his first both Eisen’saccount(1878; 1879), though incomplete, and work on Siberian enchytraeids (1973a, see p. 481) was aware Michaelsen’s(1889) revision of the types call for more affin- of H. irkutensis, but he knew only a brief and erroneous ities with the species observed by us in the Siberian samples, diagnosis provided by Cekanovskaja (1962) (seminal vesicles than with the admittedly variable European complex H. and spermathecal ectal glands omitted; spermathecal openings nasuta sensu Nielsen and Christensen (1959). described as ventral), therefore he did not consider that name as a possible identification of his own material from the shores of River Angara, material which he left unidentified as Taxonomic notes on H. ochracea “Enchytraeidae sp” (Nurminen 1973a). Later, Nurminen must have read the original work by Burov (1929), which described Nurminen (1973b, Table 2), following “the modern concepts the large seminal vesicles and the crown of 6–8 ectal glands of enchytraeid taxonomy in the sense of Nielsen and surrounding the lateral spermathecal pores (see Suppl. Table 1 Christensen (1959)“, lumped many Nordic species of in this paper), and in 1980 he reclassified Burov’s species and Henlea into synonymy with H. ochracea (A. tenellus Eisen, his own unnamed 1973 Bajkal material (in spite of the smaller 1878, H. arctica Welch, 1919, H. moderata, H. tubulifera, Hg. body size) (Nurminen 1980: p. 176 and table 1) as birulae Burov, 1929, Hg. sibiricus), but we agree with Punahenlea irkutensis. Schmelz and Collado (2012: 68, 74) Schmelz and Collado (2012) that these synonymies are in misunderstood the new combination for a new (homonymous) need of reappraisal. For instance, Nurminen recorded H. species, and renamed Nurminen’smaterialasHenlea ochracea from a mixed forest in the vicinity of Lake Bajkal irkutiana. as well as from the Canadian Arctic, but his definition of the taxon (Nurminen 1973b), white in colour and with unbranched oesophageal appendages, indicates that he dealt Conclusions with a worm distinct from that described by Eisen (1878, 1879) and augmented by Welch (1919). Furthermore, Early records of bioluminescence in Henlea were confined to Christensen and Dózsa-Farkas (1999) established H. worms of uncertain identity observed in anthropic environ- conchifera on material from the Siberian tundra, and they then ments (city gardens, greenhouses) in Europe. Our study doc- recorded it from Alaska (Dózsa-Farkas and Christensen 2002) uments that H. petushkovi sp. n. and H. rodionovae sp. n. are and widely from the Canadian Archipelago (Christensen and two native Siberian species, closely related to each other and Dózsa-Farkas 2006). From the same territories they also re- distant from all known congeners so far barcoded. Limited corded H. ochracea sensu Nurminen (see also Dózsa-Farkas hybridization may occur between them, as suggested by the et al. 2015). Having re-investigated material identified by limited conflict between COI and morphology. Our recogni- Nurminen as H. ochracea (from the Zoological Museum, tion of the polyphyletic nature of Hepatogaster and Two new bioluminescent Henlea (Clitellata, Enchytraeidae) from Siberia 311 commentaries on the status of H. nasuta, H. ochracea and H. Clement, M., Snell, Q., Walke, P., Posada, D., & Crandall, K. (2002). irkutensis,indicatethatHenlea taxonomy is still in a state of TCS: estimating gene genealogies. Proceedings of the 16th International Parallel and Distributed Processing Symposium, vol. flux, not only as concerns species interrelationships but also 2, p. 184. https://doi.org/10.1109/IPDPS.2002.1016585. with regard to species definitions. de Queiroz, K. 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