Phylogeny and Biogeography of Spinicaudata (Crustacea: Branchiopoda) Author Schwentner, Martin Center of Natural History, Universität Hamburg, Hamburg, Germany. * Correspondence: E-mail: martin. schwentner @ nhm-wien. ac. at (Schwentner) & Naturhistorisches Museum, Vienna, Austria martin.schwentner@nhm-wien.ac.at Author Rabet, Nicolas Sorbonne Université, Muséum national d’Histoire naturelle, Biologie des organismes et écosystèmes aquatiques (BOREA), CNRS, IRD, Université de Caen Basse-Normandie, CP 26 75231, 43 rue Cuvier Paris Cedex 05, France. E-mail: nicolas. rabet @ mnhn. fr (Rabet), celine. bonillo @ mnhn. fr (Bonillo) nicolas.rabet@mnhn.fr Author Richter, Stefan Allgemeine und Spezielle Zoologie, Universität Rostock, Rostock, Germany. E-mail: stefan. richter @ uni-rostock. de (Richter) stefan.richter@uni-rostock.de Author Giribet, Gonzalo Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA. E-mail: ggiribet @ g. harvard. edu (Giribet) ggiribet@g.harvard.edu Author Padhye, Sameer Systematics, Ecology & Conservation Lab, Zoo Outreach Organization, Coimbatore, Tamil Nadu, India. E-mail: sameer. m. padhye @ gmail. com (Padhye) sameer.m.padhye@gmail.com Author Cart, Jean- François 15 Avenue du Général de Gaulle, 10400 Nogent-sur-Seine, France. Email: jfcart 1 @ gmail. com (Cart) jfcart1@gmail.com Author Bonillo, Céline Sorbonne Université, Muséum national d’Histoire naturelle, Biologie des organismes et écosystèmes aquatiques (BOREA), CNRS, IRD, Université de Caen Basse-Normandie, CP 26 75231, 43 rue Cuvier Paris Cedex 05, France. E-mail: nicolas. rabet @ mnhn. fr (Rabet), celine. bonillo @ mnhn. fr (Bonillo) nicolas.rabet@mnhn.fr Author Rogers, D. Christopher Kansas Biological Survey, and The Biodiversity Institute, The University of Kansas, Higuchi Hall, 2101 Constant Avenue, Lawrence, text Zoological Studies 2020 Zool. Stud. 2020-08-05 59 44 1 23 http://dx.doi.org/10.5281/zenodo.12822688 journal article 10.6620/ZS.2020.59-44 1810-522X PMC7746975 33365101 12822648 Eocyzicus The phylogenetic relationships within Eocyzicus were not consistently resolved ( Figs. 2 , 3A , Figs. S7– S 9 ). In particular, with regards to the positions of the North American E. digueti (Richard, 1895) and the South African Eocyzicus species. While the analyses with Matrices 1 and 2 mainly suggested the latter to be sister group to all other Eocyzicus species (potentially together with Asian, North African and Middle Eastern species) ( Fig. 2 , Figs. S1–S 3 ), analyses based on Matrices 5 and 6 mainly placed E. digueti as sister group to all other Eocyzicus species ( Fig. 3A , Fig. S7– S 9 ). Below, we focus on analyses of Matrices 5 and 6 as these were tailored specifically to improve the resolution within this taxon. All Australian Eocyzicus species constitute a well supported monophyletic group (pp = 1.0; bs = 94%) with an inferred age of 52.8 mya (39.2– 67.2 mya 95% HPD) ( Fig. 4 ). This Australian clade is sister group to a clade comprising the African, Asian and Middle Eastern species (pp = 0.96–0.97; bs = 50%; RAxML analysis of Matrix 6 had suggested diverging relationships but with extremely low support). In the latter clade, E. mongolianus Uéno, 1927 ( Mongolia ) and E. orientalis Daday, 1913 ( China ) are nested within a group of Middle Eastern and South African species (pp = 1.0; bs = 89–91%). The Taiwanese species is either sister group to all of the other species of this clade or closer to the Australian clade, but with low support. More taxa are needed to better resolve this clade. The oldest divergence within Eocyzicus was dated to 95.6 mya (71.1–119.3 mya 95% HPD) but the molecular clock analyses did not include the North American E. digueti and inferred slightly different relationships among species ( Fig. 4 ). Fig. 3. Phylogenetic relationships of selected spinicaudatan taxa based on COI , 16S rRNA , EF1α and 28S rRNA inferred with MrBayes using taxonspecific matrices. A) Eocyzicidae fam. nov. and Leptestheriidae using Matrix 5 rooted based in the split between Eocyzicidae and Leptestehriidae and B) Cyzicidae s.s. using Matrix 3 rooted by the deepest split recovered in the analyses of all Spinicaudata . All individuals of the selected taxa and all codon positions were included. Genus affiliations are indicated. For Eocyzicus fam. nov. and Cyzicidae s.s. head shapes (rostrum shape and condyle length) corresponding to the four traditional cyzicid genera ( Cyzicus , Caenestheria , Caenestheriella and Eocyzicus ) are mapped based on the observed morphology of studied specimens. Rostrum shapes were differentiated into triangular and spatulated, irrespective of the presence of an additional posterior margin in the latter. Published information on the respective species was not considered to avoid errors for example due to cryptic species or wrong identifications. In some species, including E. taiwanensis , changes in rostrum morphology during growth has been observed ( e.g. , Rogers et al. 2017 ). Dotted lines indicate groups of specimen with same head shapes. Posterior probabilities and bootstrap support values are provided for each branch. Branches are color-coded according the geographic origin of the specimens. # = node in topology with highest likelihood, but bootstrap support <50%, - = node not recovered in most topology with highest likelihood. Leptestheriidae The phylogenetic relationships within Leptestheriidae were not consistently recovered among analyses ( Figs. 2–4 , Figs. S1–S 3 , S 7–S 9 , S 12–S 14). The obtained topologies of the analyses with both Leptestheriidae-specific matrices (Matrices 5 and 6) are rather similar (apart from the taxa not included in Matrix 5), with distinct differences between maximum likelihood and Bayesian analyses. The topologies obtained with the spinicaudatan-wide matrices (Matrices 1 and 2) or in the molecular clock analyses with BEAST differed markedly from these and from each other and had relatively low support for many recovered groups. Also the RAxML analyses had relatively low support values among clades. Below, we focus on analyses of Matrices 5 and 6 as these were tailored specifically to improve the resolution within this taxon. The age of extant Leptestheriidae was estimated at 126 mya (103.3–148.6 mya 95% HPD) ( Fig. 4 ). The most important difference between Matrices 5 and 6 is the presence of a European representative of Eoleptestheria ticinensis (Balsamo-Crivelli, 1859) in Matrix 5. This specimen clustered with Maghrebestheria maroccana Thiery, 1988 , though it should be kept in mind that only the relatively conserved 28S rRNA was available for the European E. ticinensis . This clade (or only M. maroccana in the analysis of Matrix 6) was sister group to all other Leptestheriidae in the Bayesian analyses (pp = 1.0; Fig. 3A , Fig. S9 ) or most other Leptestheriidae except for L. kawachiensis and an Indian species (specimen M089) in the maximum likelihood analyses ( Figs. S7 , S 8 ). The Australian specimen of E. ticinensis ( Fig. 1J ) does not cluster with the European representative, but is nested deep within Leptestheria . The uncorrected p -distance for the conservative 28S rRNA is 3.9% between these two specimens of Eoleptestheria . Leptestheria nobilis , which was formerly assigned to the now synonymized genus Leptestheriella , is also nested within a group of Leptestheria species. The leptestherid species do not group according to their geographic origin ( Figs. 2 , 3A ). Notably, there are several clusters of species from continents that once formed Gondwana, for example L. brevirostris Barnard, 1924 ( Botswana ), one specimen identified as L. rubridgei ( Baird, 1862 ) ( South Africa ), Leptestheria sp. ( Madagascar ; specimen M124) and Leptestheria nobilis ( India ) or L. venezuelica Daday, 1913 (South America), Leptestheria sp. from Brazil (South America), Leptestheria sp. ( India ; specimen M128) and Leptestheria sp. ( Madagascar ; specimen M052) (age estimated at 94 mya excluding the Madagascan species) or E. ticinensis ( Australia ), L. rubidgei ( South Africa ) and L. cortieri ( Mauritania ) with the European L. dahalacensis nested well within this last cluster (each cluster with pp = 1; Fig. 3A , Fig. S3 ). There were some instances of putatively cryptic or unrecognized species diversity within Leptestheriidae , in addition to Eoleptestheria (see above). Specimens identified as L. rubidgei were divided into two strongly separated groups (uncorrected p -distance for 16S rRNA : 4.7–5.0%) that clustered at very different positions within Leptestheriidae ( Figs. 2 , 3A ). The Algerian and Tunisian specimens of L. mayeti (Simon, 1885) differed by 12.9% uncorrected p -distance in COI from each other. In North America, L. compleximanus ( Packard, 1877 ) is currently the only recognized species. Within this species uncorrected p -distances of up to 4.8% for COI , 3.1% for 16S rRNA and 1.1% for EF1α were observed; these increased to 14.5% ( COI ) and 2.4% ( EF1α ) if the unidentified North American Leptestheria specimens are included as well. Limnadiidae There are two well (pp = 1.0; bs = 96–99%) and consistently supported main clades within Limnadiidae : a clade of Australian endemic genera ( Limnadopsis , Paralimnadia and Australimnadia ), in which Paralimnadia is sister group to Limnadopsis and a clade of species from South America ( Metalimnadia and a putative new genus), Africa ( Gondwanalimnadia and Calalimnadia ) as well as the globally distributed genus Eulimnadia ( Figs. 2 , 4 , Figs. S1–S 3 , S 12– S 14). The relationships of the Holarctic Imnadia and Limnadia species , Imnadia yeyetta Hertzog, 1935 (Europe), Limnadia lenticularis ( Linnaeus, 1761 ) (Europe), L. nipponica Ishikawa , 1895 ( Japan ) and L. americana Morse, 1868 ( USA ), are not consistently resolved ( Figs. 2 , 4 , Figs. S1–S 3 , S 12–S 14). Either Imnadia and Limnadia constituted a monophyletic clade that was sister group to the Australian clade, all other Limnadiidae , or Limnadia was the sole sister taxon to the Australian clade. However, the support values for these alternative relationships were low and nonsignificant. Fig. 4. Molecular clock dated phylogeny. The divergence time estimates are based on the BEAST analyses of COI , 16S rRNA , EF1α and 28S rRNA that included only individuals with at least three of the loci present. Blue bars represent 95% HPD intervals of inferred node ages. The topology was not constrained to enforce a sister group relationship of Leptestheriidae and Eocyzicus , thus also no prior was defined for their divergence (Fig. S12 for the constrained analysis). Calibration points (6) and (11) are based on fossils for Diplostraca (minimum age 386.9 mya based on Leaia chinensis following Wolfe et al. (2016)) and Limnadiidae + Eocyzicus + Leptestheriidae (minimum age 255 mya based on oldest known Perilimnadiidae fossils) following Astrop and Hegna (2015) , respectively (Figs. S13 and S14 for an alternative age constraint for Limnadiidae + Eocyzicus + Leptestheriidae ). Calibrations points (A–D) were inferred from the preceding molecular clock analyses of the amino acid data set of Schwentner et al. (2018) (Figs. S10, S11) and coded as normal distributed priors: (A) 294.6 mya with sigma of 25, (B) 153.5 mya with sigma of 63, (C) 66.8 mya with a sigma of 40 and (D) 64.8 mya with a sigma of 32. Branches are color-coded according the geographic origin of the specimens. Posterior probabilities are provided for each node. The divergence between the two main clades was dated to 154 mya in the PhyloBayes analyses ( Figs. S4 , S 5 ; unfortunately, no representative of Imnadia or Limnadia was available for this molecular clock analyses) and to 205.5 mya (172.2–240.2 mya 95% HPD) in the BEAST analyses ( Fig. 4 ). The age of the Australian clade was estimated to 130.3 mya (101.6–159.4 mya 95% HPD), the divergence between Paralimnadia and Limnadopsis to 105.6 mya (83.4–129.1 mya 95% HPD) (59 mya in the PhyloBayes analyses) and the oldest divergence within Limnadopsis to 65.5 mya (47.7–87 mya 95% HPD) (24 mya in the PhyloBayes analyses) ( Fig. 4 ). The split between the new South American limnadiid genus and Eulimnadia was dated to about 109.4 mya (73–145.5 mya 95% HPD) while the split between the North American Eulimnadia texana Packard, 1871 and the other Eulimnadia species studied herein was dated to 52.2 mya (36.7–70.3 mya 95% HPD).