Phylogene)c inves)ga)on of the Baikalodrilus species flock (Clitellata, Naididae) endemic to Lake Baikal, Siberia Patrick Mar)n 1 , Gontran Sonet 2 , Nathalie Smitz 3 , Sophie Gombeer 4 , Thierry Backeljau 1,2,4,5 411 Abstract Background: Lake Baikal is the deepest and most voluminous lake in the world, with a unique environment (ultraoligotrophic and well-oxygenated waters at all depths), and is located in a region that is experiencing rapid climate change. It is populated with an endemic genus of oligochaetes (Baikalodrilus), which currently comprises 21 morphospecies. However, the validity of many species is ques)onable; the great similarity in their descrip)on and the lack of unequivocal diagnos)c characters oTen lead species iden)fica)on to an impasse. In order to clarify the systema)cs of this genus, we analysed one nuclear (ITS) and two mitochondrial (COI and 16S) markers of 33 Baikalodrilus specimens and four specimens of the genera Spirosperma, Embolocephalus, Rhyacodrilus and Haber as an outgroup. Results: Phylogene)c inferences based on parsimony, maximum likelihood and Bayesian analyses showed an early separa)on between two groups of species that belong to two dis)nct size classes and helped to re-evaluate the validity of some morphological characters as specific diagnos)c characters. Three species iden)fied prior to molecular analyses were consistent with clustering based on DNA sequences. A fourth morphospecies proved to be actually an assemblage of two dis)nct species. It was also possible to isolate a group of specimens that could be considered as a new species. Other clusters remained ambiguous, not only in terms of molecular clustering but also of morphological dis)nctness. Significance: These results will be useful for a taxonomical revision of the genus and a be[er assessment of the oligochaete species diversity in a lake environment facing contemporary clima)c changes. The Baikalodrilus species flock Baikalodrilus is an endemic genus of oligochaetes to Lake Baikal, which currently comprises 21 morphospecies. By its monophyly, endemicity, and speciosity, the genus can be considered as a “species flock”. However, the validity of many species is ques)onable; the great similarity in their descrip)on and the lack of unequivocal diagnos)c characters oTen lead species iden)fica)on to an impasse. Evolu=onary history Phylogene)c inferences showed an early separa)on between two groups of species that belong to two dis)nct size classes (see B. digitatus and B. werestschagini vs. all others). The gene)c variability within the flock is low, sugges)ng a “recent” radia)on of Baikalodrilus, possibly contemporary with drama)c clima)c changes in Baikal environment that occurred at the beginning of the Pleistocene (± 2 Mya). Phylogene=c inferences In order to clarify the systema)cs of this genus, phylogene)c analyses were carried out using one nuclear (ITS) and two mitochondrial (COI and 16S) markers, and complementary phylogene)c reconstruc)ons methods (parsimony, maximum likelihood and Bayesian analyses). The three methods gave congruent results. Species delinea=on Three species iden)fied prior to molecular analyses were consistent with clustering based on DNA sequences. A fourth morphospecies proved to be actually an assemblage of two dis)nct species. It was also possible to isolate a group of specimens that could be considered as a new species. Other clusters remained ambiguous, not only in terms of molecular clustering but also of morphological dis)nctness. Morphological characters proved to be of variable usefulness as species diagnos)c characters. A taxonomical revision is badly needed but most types are seemingly lost… so that the validity of many species is queried (species inquirenda). ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( 55N 110E 105E 00.285.02 01.036.01 00.285.01 00.350.04 00.346.05 01.076.06 00.346.01 01.076.05 01.312.06 01.036.03 01.076.01 01.036.08 00.285.05 01.036.06 01.076.02 01.076.09 01.076.04 00.346.03 01.076.07 00.346.02 01.076.08 01.076.03 00.313.07 ± 0 100 200 50 Kilometers Group ! ( 1 ! ( 2 ! ( 3 ! ( 4 ! ( 5 ! ( 6 ! ( 7 ! ( 8 ! ( 9 ! ( 10 bathymetry (250 m interval) orography (500 m interval) ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( ! ( 00.350.04 00.346.05 00.346.01 01.036.03 01.065.09 00.334.02 00.165.02 00.334.01 01.065.03 01.036.09 01.076.02 01.065.02 00.313.07 ! ( ! ( ! ( ! ( ! ( ! ( 01.076.05 01.312.06 01.036.06 01.076.04 00.346.03 01.065.04 Lake Baikal is the deepest (1,637 m), the most voluminous (23,000 km 3 ) and the most ancient (25 – 30 Myr) lake in the world, with a unique environment (ultra-oligotrophic and well-oxygenated waters at all depths). It is located in a region that is experiencing rapid climate change. The lake harbours about 192 oligochaete species, of which more than 70% are endemic and species flocks are recognizable or suspected. A unique environment facing contemporary clima=c changes FIGURE 1: Phylogene)c tree based on the concatena)on of COI, 16S and 18S-ITS1-5.8S-ITS2-28S and reconstructed using the maximum likelihood method with 1000 bootstrap pseudoreplicates. 100.0 PA0905102-Papillate juvenile EY0805112-Embolocephalus yamaguchii 0228503-Embolocephalus nikolskyi 73 86 86 49 100 85 100 49 60 100 100 47 63 87 47 93 97 83 100 50 100 100 67 84 100 0016502 0028505-B_discolor 0034602 0034604 0107604 0035004-B_digitatus 0103608-B_discolor 0033401-B_malevici 0103601-B_werestschagini 0106502 0107606-B_discolor 0103609 0107608 0016104 0028502-B_cf_digitatus 0107603 0033402 0034605-B_digitatus 0131206 0031307 0107609 0028501-B_werestschagini 0106509 0107607 0034601-B_discolor 0034603 0103603 0106504 0103606-B_malevici 0107602 0016101 0107601 0107605 100 60 86 97 100 100 95 42 40 81 Affilia)ons 1 Taxonomy and Phylogeny, Royal Belgian InsEtute of Natural Sciences, rue VauEer 29, 1000 Brussels, Belgium 2 JEMU, Royal Belgian InsEtute of Natural Sciences, rue VauEer 29, 1000 Brussels, Belgium 3 JEMU/BopCo, Royal Museum of Central Africa, Leuvensesteenweg 11-17, 3080 Tervuren, Belgium 4 BopCo, Royal Belgian InsEtute of Natural Sciences, rue VauEer 29, 1000 Brussels, Belgium 5 EvoluEonary Ecology Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium FIGURE 2: Loca)on where individuals were sampled. Colour codes are used in order to match loca)ons and highlighted clusters of Fig. 1