Martin Schwentner
email: martin.schwentner@nhm-wien.ac.at

Nicolas Rabet
email: nicolas.rabet@mnhn.fr

Stefan Richter
email: stefan.richter@uni-rostock.de

Gonzalo Giribet
email: ggiribet@g.harvard.edu

Sameer Padhye
email: sameer.m.padhye@gmail.com

Jean-François Cart
email: jfcart1@gmail.com

Céline Bonillo
email: celine.bonillo@mnhn.fr

Christopher Rogers

Jean- François Cart

D Christopher Rogers

Thomas A Hegna

Special Issue: Fossil and Modern Clam Shrimp (Branchiopoda: Spinicaudata, Laevicaudata

Keywords: Spinicaudata, Laevicaudata) Phylogeny and Biogeography Clam shrimp, Caenestheria, Caenestheriella, Eocyzicidae, Gondwana, Vicariance

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BACKGROUND

Spinicaudata (spiny clam shrimp) has the most confused taxonomy of any branchiopod group due to the tremendous morphological intraspecific variability, which often overlaps with what has been considered interspecific variation, a high number of hermaphroditic lineages, and poor and inadequate descriptions and type Fig. 1. Figure 1. Representative Spinicaudata and their typical head shapes. A) Ozestheria altus [START_REF] Shu | Two new species of clam shrimp (Branchiopoda: Spinicaudata) from Yunnan Province, China[END_REF], male head left lateral view; B) Cyzicus californicus [START_REF] Packard | Synopsis of the freshwater Phyllopod Crustacea of North America[END_REF], female head left lateral view; C) Ozestheria pilosa [START_REF] Rogers | Current knowledge of the South East Asian large branchiopod Crustacea (Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida)[END_REF], male head left lateral view; D) Leptestheria kunmingensis [START_REF] Shu | Two new species of clam shrimp (Branchiopoda: Spinicaudata) from Yunnan Province, China[END_REF], male head left lateral view; E) L. kunmingensis [START_REF] Shu | Two new species of clam shrimp (Branchiopoda: Spinicaudata) from Yunnan Province, China[END_REF], female head left lateral view; F) Eocyzicus taiwanensis [START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF], male head left lateral view; G) Ozestheria sp. "Mongolia", DCR collection 729, male head left lateral view; H) Ozestheria sp. "Mongolia", DCR collection 729, male limb I endopod distal portion, right lateral view; I) Metalimnadia sp. DCR collection 853, male head left lateral view; J) Eoleptestheria cf. ticinensis from Australia, male head left lateral view. Designations: f = fornix; on = occipital notch; oc = occipital condyle; rs = rostral spine; r = rostrum. page 2 of 23 Zoological Studies 59:44 (2020) Daday's (1913aDaday's ( b 1914[START_REF] Daday | Monographie Systématique des Phyllopodes Conchostracés[END_REF][START_REF] Daday | Monographie systématique des Phyllopodes Conchostracés. II. Leptestheriidae[END_REF][START_REF] Daday | Monographie systématique des Phyllopodes Conchostracés. III. Limnadiidae[END_REF][START_REF] Daday | Monographie systématique des Phyllopodous Conchostracés. III. Limnadiidae (suite)[END_REF] monographs were criticized by many workers (e.g., [START_REF] Uéno | On some freshwater branchiopods of China[END_REF][START_REF] Barnard | Contributions to the crustacean fauna of South Africa, 10. Revision of Branchiopoda[END_REF][START_REF] Brehm | Phyllopoden. Mitteilungen von der Wallacea-Expedition Woltereck, 5[END_REF][START_REF] Gauthier | Note sur certains Conchostracés de l'Algérie et de la Tunisie[END_REF][START_REF] Linder | Affinities within the Branchiopoda with notes on some dubious fossils[END_REF][START_REF] Botnariuc | Révision des Phyllopodes Conchostracés de la collection du Professeur I. Borcea[END_REF][START_REF] Botnariuc | Contribution à la connaissance des Phyllopodes Conchostracés de Roumanie[END_REF][START_REF] Margalef | Los crustáceos de las aguas continentales ibéricas[END_REF]Straškraba 1965aStraškraba 1965b[START_REF] Straškraba | Taxonomical studies on Czechoslovak Conchostraca III. Family Leptestheriidae, with some remarks on the variability and distribution of Conchostraca and a key to the Middle-European species[END_REF], and many authors [START_REF] Vecchi | Nuova specie di Concostraco di Cirenaica[END_REF][START_REF] Gauthier | Note sur certains Conchostracés de l'Algérie et de la Tunisie[END_REF][START_REF] Linder | Affinities within the Branchiopoda with notes on some dubious fossils[END_REF][START_REF] Botnariuc | Révision des Phyllopodes Conchostracés de la collection du Professeur I. Borcea[END_REF][START_REF] Botnariuc | Contribution à la connaissance des Phyllopodes Conchostracés de Roumanie[END_REF]Straškraba 1965aStraškraba 1965b[START_REF] Straškraba | Taxonomical studies on Czechoslovak Conchostraca III. Family Leptestheriidae, with some remarks on the variability and distribution of Conchostraca and a key to the Middle-European species[END_REF][START_REF] Wiltshire | The developmental morphology of Cyzicus morsei (Packard) (Crustacea: Conchostraca) from hatching through adulthood with comments on taxonomy within the family Cyzicidae[END_REF][START_REF] Marinček | Taxonomical study of the genus Leptestheria (Conchostraca, Crustacea) I[END_REF][START_REF] Rogers | Revision of the extant genera of Limnadiidae (Branchiopoda: Spinicaudata)[END_REF][START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF] demonstrated that certain characters used to define spinicaudatan genera and species were age or food quality dependent.

The genera Caenestheria and Caenestheriella, created by Daday (1913a), have been particularly contentious. Caenestheria and Caenestheriella were separated by [START_REF] Daday | Monographie systématique des Phyllopodes Conchostracés. I. Caenestheriidae[END_REF] from Eocyzicus and Cyzicus, respectively, by the shape of the rostrum. In the former genera, the rostrum was subacute or triangular in both sexes, in the latter two genera the male rostrum was broadly spatulate. Furthermore, Cyzicus and Caenestheriella have a long, elongated occipital condyle with a narrow occipital notch, whereas Eocyzicus and Caenestheria have a short, rounded condyle forming a wide occipital notch. However, several authors synonymised Caenestheria into Eocyzicus and Caenestheriella into Cyzicus, based on developmental studies (e.g., [START_REF] Margalef | Los crustáceos de las aguas continentales ibéricas[END_REF]Straškraba 1965b;[START_REF] Wiltshire | The developmental morphology of Cyzicus morsei (Packard) (Crustacea: Conchostraca) from hatching through adulthood with comments on taxonomy within the family Cyzicidae[END_REF][START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF], demonstrating that the form of the male rostrum changed with the age of the animal. Regardless of the literature demonstrating that the definitions for these genera were problematic, the names Caenestheria and Caenestheriella continued in use (e.g., [START_REF] Tiwari | New species of Conchostraca (Crustacea: Phyllopoda) from Rajastan[END_REF][START_REF] Smith | The discovery of Caenestheriella gynecia Mattox, 1950 (Branchiopoda, Cyzicidae) in New England, with ecological and systematic notes[END_REF][START_REF] Timms | A preliminary analysis of the conchostracans (Crustacea: Spinicaudata and Laevicaudata) of the middle Paroo catchment of the Australian arid-zone[END_REF][START_REF] Stoicescu | Caenestheriella variabilis (Daday) (Conchostraca: Crustacea) a species new to the faune of Romania and its validity[END_REF][START_REF] Olesen | Caenestheriella mariae sp. nov. (Crustacea: Branchiopoda: Spinicaudata: Cyzicidae): a new clam shrimp from Western Australia[END_REF][START_REF] Richter | A list of the recent clam shrimps (Crustacea: Laevicaudata, Spinicaudata, Cyclestherida) of Australia, including a description of a new species of Eocyzicus[END_REF][START_REF] Schmidt | State records and habitat of clam shrimp, Caenestheriella gynecia (Crustacea: Conchostraca), in New York and New Jersey[END_REF]. Furthermore, [START_REF] Naganawa | Current prospect of the recent large branchiopodan fauna of East Asia: 3. revised classification of the recent Spinicaudata[END_REF] treats Eocyzicus as a synonym of Cyzicus, with no explanation or citations, an approach not followed by subsequent authors.

In Leptestheriidae, Daday's genus Leptestheriella was synonymised with Leptestheria by [START_REF] Brtek | Checklist of the valid and invalid names of the "large branchiopods" (Anostraca, Notostraca, Spinicaudata and Laevicaudata), with a survey of the taxonomy of all Branchiopoda[END_REF]. based on "… a series of changes between the two groups." However, [START_REF] Brtek | Checklist of the valid and invalid names of the "large branchiopods" (Anostraca, Notostraca, Spinicaudata and Laevicaudata), with a survey of the taxonomy of all Branchiopoda[END_REF] presented no citations, data, or explanation for this conclusion, and listed no examined material or any experiments in support.

Molecular phylogenetic studies of Spinicaudata have consistently rejected the monophyly of Cyzicidae [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF][START_REF] Sun | Phylogeny of Conchostraca based on 28S rDNA D1-D2 and partial 16S rDNA sequences[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF], with Eocyzicus (not including species originally assigned to Caenestheria or Ozestheria) being more closely related to Leptestheriidae and possibly Limnadiidae rather than with the remaining Cyzicidae (Cyzicidae sensu stricto). The species included in Cyzicidae s.s. did not cluster into the respective traditional genera but fell into two large clades. One clade included all Australian representatives that morphologically resembled Caenestheria and Caenestheriella but Schwentner et al. (2015a) showed that the two genera are not monophyletic when including the Australian representatives. The other clade included species that morphologically represented Caenestheriella and Cyzicus from North America, Japan and Europe [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF]). These results suggest that apart from Eocyzicus none of the traditional cyzicid genera are monophyletic when using molecular data and question the usefulness of the rostral shape and condyle length as genus defining characters, except for the unique combination of a short, rounded condyle and a broadly spatulate rostrum seen in Eocyzicus. However, the sampling of Cyzicidae outside of Australia was sparse in these previous studies.

The phylogenetic relationships between Eocyzicus, Leptestheriidae and Limnadiidae were not well resolved in most studies [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF]; however, phylogenomic analyses with up to 864 loci strongly supported a sister group relationship of Leptestheriidae and Eocyzicus and Limnadiidae as their closest relative [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF]). This contrasts with the morphology based hypothesis that suggested a closer relationship of Leptestheriidae and Cyzicidae [START_REF] Olesen | A phylogenetic analysis of the Conchostraca and Cladocera (Crustacea, Branchiopoda, Diplostraca[END_REF][START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF], but rather suggests that the ancestor of crown-group Spinicaudata was cyzicid-like.

Over the last few years, a number of molecular genetic studies have been published that explored the diversity and/or phylogeny within certain spinicaudatan genera, often restricted to a particular geographic region (e.g., [START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF][START_REF] Cesari | Genetic variability in European Leptestheria dahalacensis (Rüppel, 1837) (Crustacea, Branchiopoda, Spinicaudata)[END_REF][START_REF] Reed | A systematic study of the genus Eulimnadia[END_REF][START_REF] Schwentner | An integrative approach to species delineation incorporating different species concepts: a case study of Limnadopsis (Branchiopoda: Spinicaudata)[END_REF]Schwentner et al. 2012a[START_REF] Schwentner | Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure[END_REF]Schwentner et al. 2015aSchwentner et al. 2015b;;[START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF][START_REF] Weeks | The evolution of hermaphroditism from dioecy in crustaceans: selfing hermaphroditism described in a fourth spinicaudatan genus[END_REF]. Particularly for Australia, these studies revealed a much larger species diversity than expected, highlighting the need for integrative approaches to fully assess spinicaudatan diversity. These studies provided crucial insight into the diversity and local evolution of spinicaudatan taxa, but their geographic and taxonomic limitations did not allow for conclusions regarding the large-scale evolutionary or biogeographic history of Spinicaudata. All spinicaudatan families and several genera have a nearly global distribution, which could either be due to vicariance events associated with the break up and movement of continental plates or of multiple independent more recent colonization events via transcontinental and transoceanic dispersal. Only for Limnadiidae have more comprehensive analyses been published [START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF][START_REF] Weeks | The evolution of hermaphroditism from dioecy in crustaceans: selfing hermaphroditism described in a fourth spinicaudatan genus[END_REF], including a revision of its genera based on molecular phylogenetics [START_REF] Rogers | Revision of the extant genera of Limnadiidae (Branchiopoda: Spinicaudata)[END_REF]. To reconstruct the evolutionary and biogeographic history of Spinicaudata, we bring the various studies together page 3 of 23 Zoological Studies 59: 44 (2020) into a single comprehensive analysis.

MATERIALS AND METHODS

Samples, DNA extraction, PCR and sequencing

The goal was to obtain an extensive spinicaudatan dataset that brings together published sequences from various studies as well as new sequence data for species from hitherto poorly studied taxa or regions. Available spinicaudatan mitochondrial COI (cytochrome c oxidase subunit I), mitochondrial 16S rRNA, nuclear EF1α (elongation factor 1-alpha) and nuclear 28S rRNA sequences were downloaded from GenBank (Table S1). The published data had been analyzed to different extents in the associated publications. In those cases, where the available sequences had been used to delineate species using clearly stated species delineation methods (e.g., [START_REF] Schwentner | Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure[END_REF]Schwentner et al. 2015a) ) or that explicitly studied intraspecific variability (e.g., [START_REF] Cesari | Genetic variability in European Leptestheria dahalacensis (Rüppel, 1837) (Crustacea, Branchiopoda, Spinicaudata)[END_REF]) only one specimen per putative species was selected. Limnadiidae have been comprehensively studied in detail in previous studies [START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF][START_REF] Reed | A systematic study of the genus Eulimnadia[END_REF][START_REF] Rogers | Revision of the extant genera of Limnadiidae (Branchiopoda: Spinicaudata)[END_REF][START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF][START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] and especially for many Eulimnadia species only a single marker was available. Therefore, not all available data were utilized for Limnadiidae, but multiple representatives of each limnadiid genus were included (if available) (Table S1). For Cyzicidae and Leptestheriidae, the goal was also to identify potential cryptic species, therefore, as many representatives as possible were included. However, several individuals with obviously erroneous sequences (e.g., GenBank accession KF966550, which probably is an ostracod sequence) or with only a single locus available were excluded as these were problematic in preliminary analyses (e.g., when individuals of the same species did not share any loci). For Australian Eoleptestheria ticinensis and Eulimnadia sp. C loci were retrieved from published transcriptomes (GenBank BioSample SAMN06174119 and SAMN06174118; [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF]) and for Eulimnadia texana from the published genome (GenBank BioSample SAMN05965515; Baldwin-Brown et al. 2018) using local BLAST searches. For many published records species or genus names other than the currently recognized names had been used. In all our analyses we applied the currently accepted name following [START_REF] Brtek | Checklist of the valid and invalid names of the "large branchiopods" (Anostraca, Notostraca, Spinicaudata and Laevicaudata), with a survey of the taxonomy of all Branchiopoda[END_REF], [START_REF] Rogers | Revision of the extant genera of Limnadiidae (Branchiopoda: Spinicaudata)[END_REF], Schwentner et al. (2015a), Timms and Schwentner (2017), and Rogers (2020) (e.g., for most Cyzicidae s.s. this would be Cyzicus rather than Caenestheriella). We provide the name applied herein as well as the name under which the specific DNA sequences were published (Table S1).

Fresh material or soil containing viable eggs of species that were previously not studied genetically were collected in a series of field trips and expeditions performed by various authors and sometimes followed by breeding (Table S1). Because not all four markers studied herein had been sequenced in previous studies, voucher specimens or their DNA were obtained if available (e.g., from [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Schwentner | Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure[END_REF]Schwentner et al. 2015aSchwentner et al. 2015b) ) to provide additional loci for these specimens. DNA was extracted using the Qiagen Blood and Tissue or Qiagen QIAamp DNA Micro kits following the manufacturer's instructions. PCR reactions comprised 0.05 μl DreamTaq DNA Polymerase, 1.5 μl DreamTaq Buffer (Thermo Scientific), 0.12 μl dNTPs mix (25 mM each), 1.5 μl of each primer (10 mM each; see Table 1 for list of primers), and 3 μl DNA extract in a total volume of 15 μl. Temperature regimes were: 94°C for 3 min, 37 amplification cycles of 30 s at 94°C, 45 s at 46°C and 1 min at 72°C for COI; 35 amplification cycles of 30 s at 94°C, 45 s at 50°C and 1 min at 72°C for 16S rRNA; 40 amplification cycles of 30 s at 94°C, 30 s at 51°C and 1 min at 72°C for EF1α; 40 amplification cycles of 30 s at 94°C, 30s at 52.5°C and 1.5 min at 72°C for 28S rRNA and a final elongation step of 5 min at 72°C. Success of PCR reactions was assessed on 1.5% TAE gels. PCR products were cleaned-up with FastAP and Exonuclease I (both ThermoFisher Scientific). Sequencing was conducted either on an ABI Prism 3730xl Genetic Analyzer (Applied Biosystems, Carlsbad, USA) at the Bauer Core Facility at Harvard University or with Macrogen.

For Leptestheria sp. from Brazil, L. nobilis Sars, 1900 and Ozestheria sp. from Niger, DNA library preparation was performed using roughly 200 ng of extracted DNA as input. After physical shearing using a bioruptor (15 minutes, HI setting with intervalometer adjusted to 30 s ON, 30 s OFF; Diagenode, Liège, Belgium), we used a standard Ion Xpress library preparation protocol (Ion Xpress n°4471269) with enzyme concentration scaled down to 1:2. We performed the final library size selection with a double SPRI protocol using the following bead/DNA ratios: 0.55 first and then 0.25, to select fragments compatible with the 400 bp sequencing kit of the PGM platform. Sequencing libraries were multiplexed with 12 libraries of other organisms prepared in a similar fashion, using Ion Xpress barcoded adapters (Life Technologies, Carlsbad California). The equimolarity of the library pool was adjusted prior to emulsion PCR (emPCR) via a custom real-time PCR assay (SsoAdvanced supermix; Bio-Rad). No amplification of the libraries was necessary prior to emPCR. An equimolar pool of 20 pM was amplified using the One Touch2 400 bp amplification setup (ion-torrent n°4482002). The sequencing was performed on a 316v2 chip with 850 flows (400 bp sequencing kit; ion-torrent n°4479878).

For Cyzicus tetracerus from France, Ozestheria from Thailand and Madagascar, Eocyzicus saharicus [START_REF] Gauthier | Euphyllopodes et cladocérans continentaux récoltés par M. Monod au Sahara Occidental, et en Mauritanie[END_REF], Leptestheria cortieri Daday, 1913, L. dahalacensis Rüppel, 1837from Austria, L. mayeti (Simon, 1885) from Tunisia, Leptestheria spp. from India and Madagascar and the new limnadiid genus from Bolivia, library preps from DNA were performed with a Nextera XT kit (Illumina): fragmentation and Illumina adapter and index ligation. Equimolar pools of each library were established. Qualification and quantification of the final library was established before sequencing on Illumina Miseq with 2*25 Millions reads cartridge of 300 bases each (30 to 45 libraries per run).

All raw sequences were assembled and sequencing errors corrected using Geneious ® 6. 1.8 or 11.1.4. All sequences were submitted to GenBank (accession numbers MN553596-MN553672 and MN584937-MN585093; Table S1).

Alignment and phylogenetic analyses

Alignments were performed separately for each marker with MAFFT version 7 (Katoh and Standley 2013) using the slower but more thorough LINS-I option. The cyclestherid Cyclestheria hislopi (Baird, 1859) and the laevicaudatan Lynceus biformis [START_REF] Ishikawa | Phyllopod Crustacea of Japan[END_REF] were included as outgroup taxa (Table S1). The alignments of both ribosomal genes included poorly aligned regions associated with numerous indels. To avoid problems in the phylogenetic analyses due to putative erroneous aligned regions, the alignments of 16S and 28S rRNAs were masked with Zorro [START_REF] Wu | Accounting for alignment uncertainty in phylogenomics[END_REF]) and all positions with scores below 5 were removed. For the two protein-coding genes substitution saturation for codon positions 1 & 2 as well as codon position 3 was tested using the test of [START_REF] Xia | An index of substitution saturation and its application[END_REF] implemented in DAMBE6 [START_REF] Xia | DAMBE6: New tools for microbial genomics, phylogenetics and molecular evolution[END_REF]. Translated amino acid sequences were evaluated and assessed for putative stop codons. Aligned sequences for all four genes were concatenated into a single matrix. Best-fitting DNA substitution models were assessed for each gene fragment with MEGA 7 [START_REF] Kumar | MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF] following the Bayesian Information criterion (BIC).

Prior to phylogenetic analyses, the separate alignments per gene were concatenated into six different matrices. Matrix 1 included all taxa from all four gene fragments. For Matrix 2, individuals with less than two of the four loci present were excluded (to reduce the impact of missing data) and the 3rd codon position of COI removed (to reduce the impact of substitution saturation that was detected for the 3rd codon position of COI). Matrix 3 and Matrix 4 were taxon-specific matrices that included only representatives of Cyzicidae s.s. For these matrices, new taxon-specific alignments were computed for 16S and 28S rRNA. Because the species are more closely related, these alignments had hardly any indels, which greatly decreased alignment uncertainties and eliminated the need for masking. Thereby particularly variable regions were retained, which might be relevant to resolve the relationships among closely related species. Matrix 3 included all Cyzicidae s.s. and Matrix 4 only those representatives with at least two of the four loci present. Phylogenetic analyses of these two matrices were rooted by the wellsupported split between the two main clades within page 5 of 23 Zoological Studies 59:44 (2020) Cyzicidae s.s. (see below). Matrix 5 and Matrix 6 were constructed similarly as Matrices 3 and 4, but taxonspecific for Leptestheriidae and Eocyzicus. Matrix 5 included all representatives of these two taxa and Matrix 6 only those with at least two loci present. The phylogenetic analyses were rooted by the split between Eocyzicus and Leptestheriidae. The 3rd codon position of COI was not removed in any of the taxon-specific matrices as substitution saturation within these taxa was lower. No Limnadiidae-specific matrix was constructed and analysed, as no well-supported point for rooting was available.

Phylogenetic analyses were conducted with MrBayes 3.2.6 [START_REF] Ronquist | MrBayes 3.2: Efficient Bayesian phylogenetic inference and model selection across a large model space[END_REF]) and RAxML 8.2.12 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]. MrBayes analyses consisted of six runs, four chains and 30*106 generations for each of the six matrices. The first 10% of generations were discarded as burn-in, after assessing convergence with Tracer v1.6 [START_REF] Rambaut | Tracer v1.6[END_REF], and the majority rule consensus tree calculated for the remaining generations. The matrices were partitioned by gene fragment and the best-fitting evolutionary model (GTR + I + G) was applied to each partition independently. Maximum likelihood analyses with RAxML were run under the GTR + G model (-m GTRGAMMA) as it is suggested to ignore the invariant parameter in RAxML. Tree searching and bootstrapping was performed in a single run (-f a), using 500 bootstrap replicates and partitioned by genes. All MrBayes and RAxML analyses were run on the CIPRES Science Gateway. The resulting phylogenetic trees were visualized with FigTree 1.4. 3 Rambaut 20063 Rambaut -2019)).

Molecular clock analyses

Despite the overall wealth of spinicaudatan fossils, few can be safely assigned to extant spinicaudatan families let alone genera [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. To overcome these shortcomings, we used a twostep approach for the molecular clock analyses. First, we used the transcriptome-based data set with 606 genomic loci and an average taxon occupancy per locus of 84% of [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF] with several fossil calibration points across Branchiopoda. This data set includes eleven Spinicaudata and allows an overall overview of the timing of key cladogenetic events within Spinicaudata but not detailed age estimates within families or with regards to single biogeographic events. As a second step, we analyzed our speciesrich phylogenetic data set using several of the inferred divergence times within Spinicaudata from the former molecular clock analyses of the amino-acid data set as additional calibration points.

We used the amino-acid matrix (matrix 7

of [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF], which focused on Branchiopoda with one remipede (Xibalbanus tulumensis (Yager, 1987)) and one cephalocarid (Lightiella incisa Gooding, 1963) as outgroups.

The topology was fixed to the topology obtained by [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF]. Node ages were estimated with PhyloBayes 4.1 [START_REF] Lartillot | A Bayesian mixture model for acrosssite heterogeneities in the amino acid replacement process[END_REF] Astrop and Hegna 2015), as Perilimnadiidae has been suggested to be ancestral to all Limnadiidae except Limnadopsis, though [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF] questioned the exclusion of Limnadopsis.

To t e s t t h e i m p a c t o f t h e s p i n i c a u d a t a n fossil calibration point (calibration point ( 11)), the PhyloBayes analysis was repeated with the split between Limnadiidae and Eocyzicus + Leptestheriidae set to a minimum of 380 mya based on the assumption that Vertexioidea (known since the mid Devonian) is ancestral to Limnadiidae, while Eosestherioidea is ancestral to Leptestheriidae and Cyzicidae [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF].

For the subsequent molecular clock analyses with our species-rich data set, a new matrix was constructed for the four loci that included only individuals with at least three loci present and only one representative per species. This second molecular clock analysis was run in BEAST v2.4.6 [START_REF] Bouckaert | BEAST 2: A software platform for Bayesian evolutionary analysis[END_REF]) as here normal distributions for node ages inferred from the previous molecular clock analysis could be more precisely defined than in PhyloBayes. Tree models were linked across all loci and site and clock models were unlinked. The GTR model with invariant sites and a relaxed log normal clock with a Birth Death model was applied to all partitions. Four BEAST molecular clock analyses were run, one run each using the age estimates derived from the two different fossil calibration points for the split between Limnadiidae and Eocyzicus + Leptestheriidae (see above, calibration point ( 11)). Each of these were run once with an additional age constraint on the split between Eocyzicus and Leptestheriidae (enforcing monophyly for this node) and once without this constraint (the latter accommodates the possibility that Eocyzicus and Leptestheriidae are not sister taxa). The fossil calibration points for Diplostraca (calibration point ( 6)) as well as the split between Limnadiidae and Eocyzicus + Leptestheriidae (calibration point (11)) were applied as described above with a uniform distribution. For all calibration points that were derived from inferred nodes of the first molecular clock analysis priors with a normal distribution were applied. Here the inferred mean values were coupled with a sigma that best modelled the 95% Highest Probability Density (HPD) distribution of the inferred node ages. Applied node ages were for the molecular clock analyses with the 255 mya calibration point for Limnadiidae + Leptestheriidae + Eocyzicus: (A) 294.6 mya with a sigma of 25 (403 mya with sigma 10 when 380 mya calibration point for Limnadiidae + Leptestheriidae + Eocyzicus was applied) for crown group Spinicaudata (B) 153.5 mya with sigma of 63 (155.6 mya with sigma 70) for the split between the Australian Limnadiidae and Eulimnadia clades, (C) 66.8 mya with a sigma of 40 (61.6 mya with sigma 30) for the split between Limnadopsis and Paralimnadia, (D) 64.8 mya with a sigma of 32 (67 mya with sigma 35) for the split between O. pilosa and the Australian Ozestheria species and where applicable (E) 146 mya with a sigma of 69 (154 mya with sigma 75) for the split between Eocyzicus and Leptestheriidae.

RESULTS

The alignments including all spinicaudatans for COI, 16S rRNA, EF1α and 28S rRNA were 599 bp, 449 bp, 746 bp and 675 bp, respectively. In EF1α no substitution saturation was detected, though for COI, substitution saturation was high and significant for the 3rd codon position. After removing all 3rd codon positions, the COI alignment had a length of only 400 bp. For each gene fragment, the GTR + G + I evolutionary model was inferred. No stop codons were detected in COI and EF1α.

Phylogenetic analyses recovered monophyly of Limnadiidae and Leptestheriidae, while Cyzicidae was paraphyletic. Exclusion of Eocyzicus, renders Cyzicidae monophyletic, with all the members of Eocyzicus forming an independent clade (Figs. 2,3; Figs. S1-S9). We refer to the former as Cyzicidae sensu stricto. Leptestheriidae was recovered as sister group to Limnadiidae and Eocyzicus as their closest relatives, each with full support (posterior probability [pp] = 1.0) in the Bayesian analyses, and varying poorly supported relationships among families in the RAxML analyses, but never with Eocyzicus as sister to Cyzicidae s.s. Internal relationships within families were not always consistently recovered (Figs. 234, Figs. S1-S14), and for these we focus predominantly on the results obtained with the taxon-specific analyses.

In the molecular clock analyses of the transcriptome data set with PhyloBayes, we used the topology by [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF], which proposed Eocyzicus to be sister taxon to Leptestheriidae. Here the two alternative calibration points for the split between Limnadiidae and Eocyzicus + Leptestheriidae, despite differing by 125 my, had relatively little influence on the inferred node ages within Spinicaudata. Only for this specific node (mean ages 389 mya vs. 271 mya) and the age of crown-group Spinicaudata (mean ages 403 mya vs. 295 mya) was a larger difference in the age estimates observed (Figs. S10,S11). However, in the subsequent molecular clock analyses with BEAST the age of this calibration point had a stronger effect on the inferred node ages, with differences of ~30% between analyses (Fig. 4, Figs. S12-S14). Constraining the BEAST analyses to force a sister group relationships S1), if no collection or voucher number was available one of the GenBank numbers is provided. 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. of Leptestheriidae and Eocyzicus did not affect inferred node ages (usually < 1% difference between constrained and unconstrained). In general, older node ages were inferred in the more species-rich BEAST analyses than in the preceding loci-rich PhyloBayes analyses (Fig. 4, Figs. S10-S14). In the following, we focus on the BEAST analyses with the more conservative fossil calibration point (276 mya) (Fig. 4).

Cyzicidae s.s.

Cyzicidae s.s. is divided into two well-supported clades (each pp = 1.0 and bootstrap support [bs] = 100%) that diverged about 239.1 mya (194.9-282.2 mya 95% HPD) (Figs. 234, Figs. S4-S6). One clade includes all Australian representatives (Ozestheria), which constitute a monophyletic group itself (pp = 1.0; bs = 93 and 94%), as well as species from Southeast Asia (Thailand), Central Asia (Mongolia) and Africa (Niger and Madagascar). Ozestheria pilosa (Thailand) or O. pilosa together with the Mongolian species constitute the sister group to all Australian Ozestheria species and the whole clade was dated to 151.1 mya (120-184.3 mya 95% HPD). The divergence between O. pilosa and Australian Ozestheria was dated to 119.9 mya (96.3-141.7 mya 95% HPD, Fig. 4) (only 65 mya in the PhyloBayes analyses, Figs. S10, S11), while the oldest divergence in the Australian clade was estimated to about 94.6 mya (76.2-112.6 mya 95% HPD) (36.6 in the PhyloBayes analyses). The two African species clustered together (pp = 1; bs = 100%) and were sister group to all other species of this clade (pp = 0.94-1.0; bs = 33%; Figs. 2,3,. The two African species, as well as O. pilosa, were originally assigned to Cyzicus. However, subsequent examinations revealed that they meet the morphological characterization of Ozestheria; i.e., the male claspers (= thoracopod I and thoracopod II) having the elongated, spatulate (clawlike) spines or scales in a transverse row at the endopod apex.

The second clade includes Palearctic and Nearctic species from Europe (e.g., France, Austria, Romania and Ukraine), East Asia (Japan), northern Africa (Morocco), Middle East (Jordan) and North America (USA). The North American species constitute a monophyletic group (pp = 1.0; bs = 99-100%) that diverged from all other species of this clade about 116.6 mya (86.2-148.4 mya 95% HPD) (Figs. 234). The only possible exception being one specimen identified as Cyzicus setosus [START_REF] Pearse | Notes on Phyllopod Crustacea[END_REF] from an unknown location (GenBank accessions DQ310628 and DQ310668, deWaard et al. 2006; C. setosus is a North American species), which did not cluster with other representatives of the same species but rather with the northern African, Japanese and European species. We strongly suspect that this specimen was mislabelled or misidentified.

Specimens identified as Cyzicus tetracerus (Krynicki, 1830) did not cluster together and may constitute up to four species, respectively collected in France, Ukraine, Austria/Romania and Jordan (Figs.

2, 3B, Figs. S4-S6). Uncorrected p-distances in COI exceeded 13% between each of these putative species. As C. tetracerus was first described from Kharkiv in the Ukraine, the Ukrainian population may constitute the "true" C. tetracerus. Whether the others represent species new to science or species that have been previously synonymized with C. tetracerus is currently unknown. The North American species Cyzicus setosus and Cyzicus californicus [START_REF] Packard | Synopsis of the freshwater Phyllopod Crustacea of North America[END_REF]) clustered together and share identical 16S sequences, suggesting that these could be conspecific, while the other two North American species Cyzicus mexicanus (Claus, 1860) and Cyzicus gynecius [START_REF] Mattox | Notes on the life history and description of a new species of conchostracan phyllopod, Caenestheriella gynecia[END_REF] differed by 3.7-5.5% uncorrected p-distances in 16S.

The head morphology (i.e., condyle length and rostrum shape) did not correspond to single clades recovered in the phylogenetic analyses (Fig. 3B). Rather all combinations that defined traditional genera were associated with multiple clades that were not closely related. In several instances groups of closely related species shared identical head morphologies (for example in Eocyzicus or several Ozestheria species). However, in other instances individuals that were genetically so similar that they probably constitute a single species differed in their head morphology. In O. pilosa the length of the condyle varied from short to long, whereas the genetically indistinguishable C. setosus and C. californicus differed in the shape of their rostrum. In general, specimens with the condylerostrum combination that is typical for Eocyicus (short condyle, spatulated rostrum) fell into Eocyzicus and not into Cyzicidae s.s., the only exception being Ozestheria sp. from Mongolia. This specimen had the typical head morphology of Eocyzicus but with an additional spine at the tip of the rostrum (Fig. 1).

Eocyzicus

The phylogenetic relationships within Eocyzicus were not consistently resolved (Figs. 2, 3A, Figs. S7-S9). 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 group to all other Eocyzicus species (Fig. 3A, Fig. S7-S9). 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).

Leptestheriidae

T h e p h y l o g e n e t i c r e l a t i o n s h i p s w i t h i n Leptestheriidae were not consistently recovered among analyses (Figs. 234, Figs. S1-S3, S7-S9, S12-S14). 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,S8). 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 [START_REF] Baird | Description of seven new species of phyllopodous crustaceans, belonging to the genera Estheria and Limnetis[END_REF]) (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 [START_REF] Packard | Descriptions of new phyllopod Crustacea from the west[END_REF] 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 to the Australian clade. However, the support values for these alternative relationships were low and nonsignificant.

The divergence between the two main clades was dated to 154 mya in the PhyloBayes analyses (Figs. S4, S5; 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).

DISCUSSION

Phylogenetic and Systematic Implications

The taxonomy and systematics of Spinicaudata has always been problematic due the lack of welldefined diagnostic characters and the prevalence of high intraspecific and intrageneric morphological variability [START_REF] Vecchi | Nuova specie di Concostraco di Cirenaica[END_REF][START_REF] Brehm | Phyllopoden. Mitteilungen von der Wallacea-Expedition Woltereck, 5[END_REF][START_REF] Gauthier | Note sur certains Conchostracés de l'Algérie et de la Tunisie[END_REF][START_REF] Linder | Affinities within the Branchiopoda with notes on some dubious fossils[END_REF][START_REF] Botnariuc | Révision des Phyllopodes Conchostracés de la collection du Professeur I. Borcea[END_REF][START_REF] Botnariuc | Contribution à la connaissance des Phyllopodes Conchostracés de Roumanie[END_REF][START_REF] Margalef | Los crustáceos de las aguas continentales ibéricas[END_REF]Straškraba 1965aStraškraba b 1966;;[START_REF] Wiltshire | The developmental morphology of Cyzicus morsei (Packard) (Crustacea: Conchostraca) from hatching through adulthood with comments on taxonomy within the family Cyzicidae[END_REF][START_REF] Marinček | Taxonomical study of the genus Leptestheria (Conchostraca, Crustacea) I[END_REF][START_REF] Rogers | Revision of the extant genera of Limnadiidae (Branchiopoda: Spinicaudata)[END_REF][START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF]. Previous studies had already suggested the non-monophyly of Cyzcidae s.l., with Eocyzicus being closer related to Leptestheriidae and/or Leptestheriidae + Limnadiidae [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF]. Our results strongly support this finding with the most species-rich spinicaudatan molecular data set studied so far. As a consequence, the genus Eocyzicus should be placed in its own family and the family definition of Cyzicidae s.s. needs to be adjusted accordingly (see taxonomic section below). Although our phylogenetic analyses suggest a sister group relationship of Leptestheriidae with Limnadiidae, a scenario already proposed by [START_REF] Sars | Description of two additional South African Phyllopoda[END_REF], we think that a sister group relationship of Eocyzicus with Leptestheriidae is more likely given the results of the phylogenomic study of [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF], which is based on a much larger set of molecular loci.

A l l f o u r m a i n t a x a ( = f a m i l i e s ) w i t h i n Spinicaudata-Limnadiidae, Leptestheriidae, Eocyzicidae (see below) and Cyzicidae s.s.-are unambiguously and strongly supported as monophyletic. This is particularly notable for Leptestheriidae, which were represented in previous molecular phylogenetic studies solely by species of the genus Leptestheria [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF].

Unfortunately, the phylogenetic relationships within Leptestheriidae were not well resolved, but it appears that Maghrebestheria (possibly together with the European Eoleptestheria) is the sister group to all other Leptestheriidae. The taxonomic status of Eoleptestheria is challenged, with the Australian representative nesting within Leptestheria species and the European with Maghrebestheria. There is no support for maintaining the previously synonymized genus Leptestheriella [START_REF] Brtek | Checklist of the valid and invalid names of the "large branchiopods" (Anostraca, Notostraca, Spinicaudata and Laevicaudata), with a survey of the taxonomy of all Branchiopoda[END_REF] as the only species in our study that had formerly been assigned to Leptestheriella (L. nobilis) also nests within Leptestheria. Our results suggest that the systematics of Leptestheriidae are in need of revision. However, our results also highlight the need to include more species and to better resolve their overall relationships before such revisions can and should be conducted. Therefore, we did not include Leptestheriidae in the taxonomic section of the present study. Within Cyzicidae s.s., our results suggest that the distribution of Ozestheria is extended from Australia, as proposed by Schwentner et al. (2015a), to Asia and Africa. It is probable that further cyzicids from these continents belong to Ozestheria as well. The genera Caenestheria and Caenestheriella are not supported (as previously suggested by [START_REF] Vecchi | Nuova specie di Concostraco di Cirenaica[END_REF][START_REF] Gauthier | Note sur certains Conchostracés de l'Algérie et de la Tunisie[END_REF][START_REF] Linder | Affinities within the Branchiopoda with notes on some dubious fossils[END_REF][START_REF] Botnariuc | Révision des Phyllopodes Conchostracés de la collection du Professeur I. Borcea[END_REF][START_REF] Botnariuc | Contribution à la connaissance des Phyllopodes Conchostracés de Roumanie[END_REF][START_REF] Margalef | Los crustáceos de las aguas continentales ibéricas[END_REF]Straškraba 1965b[START_REF] Straškraba | Taxonomical studies on Czechoslovak Conchostraca III. Family Leptestheriidae, with some remarks on the variability and distribution of Conchostraca and a key to the Middle-European species[END_REF][START_REF] Wiltshire | The developmental morphology of Cyzicus morsei (Packard) (Crustacea: Conchostraca) from hatching through adulthood with comments on taxonomy within the family Cyzicidae[END_REF][START_REF] Marinček | Taxonomical study of the genus Leptestheria (Conchostraca, Crustacea) I[END_REF][START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF][START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF]Schwentner et al. 2015a)), and a thorough revision of all Cyzicidae s.s. appears necessary. The length of the condyle and especially the shape of the rostrum (Figs. 1,3) are not suitable taxonomic characters to define and differentiate cyzicid genera, not even among adult specimens. Developmental studies had already shown that rostrum shape can vary with age [START_REF] Margalef | Los crustáceos de las aguas continentales ibéricas[END_REF]Straškraba 1965b;[START_REF] Wiltshire | The developmental morphology of Cyzicus morsei (Packard) (Crustacea: Conchostraca) from hatching through adulthood with comments on taxonomy within the family Cyzicidae[END_REF][START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF]. Finding new informative characters will be a crucial challenge for future taxonomic revisions of Cyzicidae.

Although the internal relationships of the limnadiid genera are also not fully resolved, in particular of Imnadia and Limnadia, at least the monophyly of the various genera appears well supported. The monophyletic group comprising the Australian genera Limnadopsis, Paralimnadia and Australimnadia had been recovered previously [START_REF] Schwentner | Phylogeny of Spinicaudata (Branchiopoda, Crustacea) based on three molecular markers -an Australian origin for Limnadopsis[END_REF][START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF] Eulimnadia, Metalimnadia, Calalimnadia and Gondwanalimnadia [START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF][START_REF] Weeks | The evolution of hermaphroditism from dioecy in crustaceans: selfing hermaphroditism described in a fourth spinicaudatan genus[END_REF][START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF]. Apparently Limnadopsis and Paralimnadia are sister taxa, which is slightly surprising given the fact that most species of Limnadopsis and Australimnadia are quite large bodied representing the largest extant Spinicaudata. Either their large body size was acquired independently or the species of Paralimnadia reduced its size secondarily.

TAXONOMY Cyzicidae Stebbing, 1910

= Caenestheriidae Daday, 1913: 12 (pro Comments: The Cyzicidae is restricted to two genera: Cyzicus and Ozestheria. Cyzicus is fixed as the type genus for the family (Daday 1913a b).

Cyzicus Audouin, 1837

= Caenestheriella [START_REF] Daday | Monographie systématique des Phyllopodes Conchostracés. I. Caenestheriidae[END_REF]: 106 fide Margalef, 1953, fide Straškraba 1965b= Caenestheria Daday, 1914: 53 valve breadth (umbone to margin). Carapace growth line intervals smooth or ornamented (scarring from algae often mistaken for ornamentation). Carapace typically dark brown, occasionally black, or with yellow markings, often with setae. Clasper endopod (= movable finger) apically unarmed, or with a few setae, apical margin crenulate at most, but never with claw-like seta or scales. Endite IV broadly transverse to cylindrical, bearing a dense, apical field of short spiniform setae. Thoracic segments smooth or with a central dorsoposterior projection and/or set of spines or setae. Eggs attaching to prolonged exopods of thoracopods IX and X. Thoracopod exopods lacking a triangular lamina. Telson posteriolateral spine rows confluent dorsally, with confluence not projecting. Each row has from 10 to 30 spines depending on species. Caudal filament (= telsonic filament) originating between spine rows at fifth, sixth, or seventh spines from confluence. Caudal filament borne or not on projecting mound. Cercopods arcuate, occasionally sinuate, or straight with distal fourth to third bent dorsally. Cercopod with medial longitudinal setal row on proximal 40-60%. Setae plumose and either long or short. Setal row terminates with single spine. Cercopod with subapical, dorsal cirrus, extending from 60-40% of cercopod length. Eggs smooth, unornamented.

Comments: The type species is Limnadia tetracera Krynicki, 1830 (now recognized as C. tetracerus) fixed by monotypy. Currently, we recognize about 25 species in this genus [START_REF] Rogers | Spinicaudata Catalogus (Crustacea: Branchiopoda)[END_REF]. At this time the genus is morphologically defined based upon the family characters and the clasper endopod apically unarmed, or with apical margin crenulate at most. Further molecular and morphological analyses are needed to clarify relationships among the members of this genus. The genera Caenestheria and Caenestheriella are not supported in our analysis. Indeed, one species in our analysis that was formerly attributed to Caenestheriella, Cyzicus setosus, appears to be to be conspecific with Cyzicus californicus.

Ozestheria Schwentner and Richter, in

Schwentner, Just, and Richter, 2015

Diagnosis: (modified from Schwentner et al. 2015). Populations composed of males and females; amplexus is venter to venter. Male and female rostrum triangular. Ocular tubercle smoothly arcuate. Angle between rostrum and frons 150° to 170°. Occipital condyle either short and rounded or elongated and subacute. Carapace valve length ~1.5 times valve breadth (hinge to margin). Carapace with or without page 14 of 23 Zoological Studies 59: 44 (2020) sculpturing between growth lines (scarring from algae often mistaken for sculpture). Carapace typically dark brown. Male thoracopod I and II with endopod (= movable finger) bearing one or more transverse apical rows of flattened, broadly subtriangular denticles (clawlike scales) (Fig. 1H). Endite IV broadly transverse to cylindrical, bearing a dense, apical field of short spiniform setae. Eggs attaching to prolonged exopods of thoracopods IX and X. Thoracopod exopods lacking a triangular lamina. Posterior trunk segments with several medial dorsoposterior spines per segment. Telson posterior margin posteriolateral spine rows confluent dorsally, with confluence not projecting. Each row with 10 to 30 spines. Caudal filament originating between spine rows at fifth, sixth, or seventh spines from confluence. Caudal filament borne on projecting mound or not. Cercopods sinuate to curved. Cercopod with medial longitudinal setal row on proximal 40-60%. Setae plumose and either long or short. Setal row terminates with single spine. Cercopod with subapical, dorsal cirrus, extending from 60-40% of cercopod length.

Comments: Ozestheria lutraria [START_REF] Brady | Notes on freshwater Entomostraca from South Australia[END_REF] is the type species (Schwentner et al. 2015). This genus is morphologically defined by the clasper endopod bearing one or more transverse apical rows of flattened, broadly subtriangular denticulae ("claw-like scales"). Several forms previously treated at Cyzicus are here reported as Ozestheria, with the result that Ozestheria is no longer considered to be endemic to Australia. The distribution of this genus apparently extends to Africa and Asia as well suggesting to be primarily Gondwanan, with an extension into Asia. It is currently unclear if further species, which are currently identified as Cyzicus, in fact belong to Ozestheria. But at this time we can confidently ascribe at least one Asian species to this genus: Ozestheria pilosa [START_REF] Rogers | Current knowledge of the South East Asian large branchiopod Crustacea (Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida)[END_REF] = Caenestheriidae Daday, 1913: 12 (pro partim) Diagnosis: (Modified from [START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF]). Populations composed of males and females; amplexus is venter to venter. Rostrum typically sexually dimorphic. Rostrum subtriangular (usually females) to subquadrate (usually males) or rounded. Adult rostrum not armed with an apical spine (sometimes present in juveniles). Angle between rostrum and frons 170° to 190°. Occipital notch very shallow or absent. Occipital condyle low, rounded or absent, length half or less basal width. Carapace valve length ~1.5 times valve breadth (hinge to margin). Carapace growth line intervals smooth or weakly ornamented (scarring from algae often mistaken for ornamentation). Carapace typically whitish and partly translucent. Clasper endopod apically with one or a few elongated scales, each scale laterally fringed. Endite IV broadly transverse to cylindrical, bearing a dense, apical field of short spiniform setae. Thoracic segments smooth or with a central dorsoposterior projection and/or set of spines or setae. Eggs attaching to prolonged exopods of thoracopods IX and X. Thoracopod epipods lacking a triangular lamella. Telson posterior margin posteriolateral spine rows confluent dorsally, with confluence not or slightly projecting. Each row has from six to 30 spines depending on species and gender. Females typically have more and smaller spines than males. Caudal filament originating between spine rows at fifth, sixth, or seventh spines from confluence. Caudal filament borne on projecting mound. Cercopods arcuate or straight. Cercopod with a dorsomedial longitudinal row of setae or spines on proximal 40-60%. Setae plumose and either long or short. Row terminates with single spine. Cercopod with subapical, dorsal cirrus, extending from 50-40% of cercopod length. Eggs smooth or with surface polygons.

Comments: The type genus is fixed here as Eocyzicus. [START_REF] Naganawa | Current prospect of the recent large branchiopodan fauna of East Asia: 3. revised classification of the recent Spinicaudata[END_REF] treated Eocyzicus as a junior synonym of Cyzicus, however. this is not supported by this and previous molecular studies (Schwenter et al. 2009(Schwenter et al. 2018;;[START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF]. = Caenestheria Daday, 1914 (pro partim) Diagnosis: As for the family.

Eocyzicus

Comments: The type species for the genus is Eocyzicus orientalis [START_REF] Daday | Monographie systématique des Phyllopodes Conchostracés. I. Caenestheriidae[END_REF], fixed here by designation. The synomyzation of Cyzicus with Eocyzicus is not supported. Species following Daday's (1913a b) description of Caenestheria are now summarized under Cyzicus (see above). Relationships within Eocyzicus remain unclear, and further sampling across many taxa is required before any meaningful relationships can be determined.

Biogeographic History

To date, little is known about the biogeographic history of extant Spinicaudata. The spinicaudatan fossil record is rich and diverse, but limited almost exclusively to carapaces. Thus the fossil record contributes little information for the evolutionary history of extant taxa page 15 of 23 Zoological Studies 59: 44 (2020) as the fossil relationships are not well resolved and still debated [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. It is worth noting that all four spinicaudatan families have a nearly global distribution (the Americas, Eurasia, Africa and Australia) and even genera such as Leptestheria, Eocyzicus, Cyzicus and Eulimnadia occur on all or most of these landmasses. Within continents, Spinicaudata have revealed remarkable dispersal potential with species being distributed over > 1000 km and relatively low levels of population differentiation [START_REF] Cesari | Genetic variability in European Leptestheria dahalacensis (Rüppel, 1837) (Crustacea, Branchiopoda, Spinicaudata)[END_REF]Schwentner et al. 2012a[START_REF] Schwentner | Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure[END_REF]Schwentner et al. 2015a)). Such longdistance dispersal can be mediated via birds or other animal vectors or wind [START_REF] Bilton | Dispersal in freshwater invertebrates[END_REF]) and probably follow the same model as described for the related Anostraca [START_REF] Rogers | A conceptual model for anostracan biogeography[END_REF] and references cited within).

Our molecular clock analyses suggest that the four extant spinicaudatan families diverged prior to the break-up of Gondwana and Laurasia and possibly of Pangea. Also the divergence between species and species groups inhabiting different continents mostly predate the separation of the respective continental plates. Historic vicariance appears to be the main factor explaining the transcontinental distribution of extant spinicaudatan taxa, though in a few instances more recent transoceanic dispersal events have occurred as well. For other large Branchiopoda, like Anostraca, Laevicaudata, and Cyclestherida, vicariance apparently also played a major role in shaping today's distribution of taxa [START_REF] Schwentner | Cyclestheria hislopi (Crustacea: Branchiopoda): a group of morphologically cryptic species with origins in the Cretaceous[END_REF][START_REF] Rogers | A conceptual model for anostracan biogeography[END_REF]Sigvardt et al. in press), in particular for southern hemisphere taxa. For Notostraca, previous molecular clock analyses obtained different estimates of their divergence times. While some suggested more recent divergence ages, implying trans-oceanic dispersal events (Mather et al. 2013;[START_REF] Vanschoenwinkel | Toward a global phylogeny of the "living fossil" crustacean order of the Notostraca[END_REF], others suggested divergence times that imply vicariance events [START_REF] Korn | Molecular phylogeny of the Notostraca[END_REF]. Our own divergence time estimates within Notostraca (Figs. S10,S11) are similar to the former. One should keep in mind that we predominantly discuss our molecular clock results based on the more conservative calibration point of 255 mya for Limnadiidae + Leptestheriidae + Eocyzicus, the divergence ages inferred using the 380 mya calibration point were even older (Figs. S13-S14) and thus even stronger in suggesting vicariance over dispersal.

The divergence between northern and southern hemisphere species of Cyzicidae and Limnadiidae (with the exception of Eulimnadia) probably predates the break-up of Pangea in the early to mid-Jurassic, suggesting that geographic distances might have separated these clades already on Pangea. If Limnadia and Imnadia are indeed nested among the two southern continental clades, a southern hemisphere origin can be hypothesized for Limnadiidae. From there the northern hemisphere could have been colonized when Gondwana and Laurasia were still joined in Pangea. Also for Leptestheriidae, a southern hemisphere origin can be assumed, based on the phylogenetic relationships among its taxa. But here multiple independent colonization events of northern hemisphere continents have to be assumed. The strongly diverging relationships within Leptestheriidae among most analyses do not allow establishing detailed biogeographic hypotheses for this taxon. Most inferred clades within Leptestheriidae do not correspond to geographic regions (e.g., African or Indian species do not form monophyletic groups each) and many deep splits within Leptestheriidae were dated to be 90 mya or older, which roughly coincides with the separation between Africa and South America (~100 mya; McLoughlin 2001) and predates the final break-off of Australia (~50 mya; [START_REF] Beaulieu | A Southern Hemisphere origin for campanulid angiosperms, with traces of the break-up of Gondwana[END_REF]. Ancestral leptestheriid lineages were probably once widely distributed across Gondwana and when Gondwana broke apart, several of these lineages survived on more than one continent. For example, one leptestheriid clade that was consistently recovered comprised Leptestheria nobilis from India, Leptestheria sp. (specimen M124) from Madagascar and L. brevirostris from Botswana with an estimated clade age of 95 mya. A Madagascar-India-Seychelles block was the first landmass that broke-off from Gondwana around 120 mya [START_REF] Ali | Gondwana to Asia: Plate tectonics, paleogeography and the biological connectivity of the Indian sub-continent from the Middle Jurassic through latest Eocene (166-35 Ma)[END_REF]Aitchinson 2008, McLoughlin 2001) and this leptestheriid clade probably evolved on this landmass and dispersed from Madagascar to continental Africa subsequently. Clades with similar African and Indian distributions were also recovered for the notostracan Triops (e.g., clades 18 and 26 in [START_REF] Korn | Molecular phylogeny of the Notostraca[END_REF]; see also [START_REF] Modak | Molecular phylogenetic investigations of Triops granarius (Lucas, 1864) (Branchiopoda: Notostraca) from the type locality of the former Apus orientalis Tiwari, 1952 and three other localities in the Western Ghats of India[END_REF]. While some of these distributions might be due to the geological processes (e.g., clade 18 in [START_REF] Korn | Molecular phylogeny of the Notostraca[END_REF], others appear to be younger and thus possibly due to more recent dispersal and colonization events (e.g., clade 26 in [START_REF] Korn | Molecular phylogeny of the Notostraca[END_REF]).

The only spinicaudatan example of repeated transoceanic dispersal is Eulimnadia. This genus probably evolved in South America and successfully dispersed to virtually all other continents (there are no extant records from Antarctica, whether Antarctica was historically inhabited is unknown), as well as many oceanic islands [START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF], probably during the last 50 mya. [START_REF] Bellec | Dating the Limnadiidae family suggests an American origin of Eulimnadia[END_REF] dated the onset of this global distribution to only 30 mya. Eulimnadia is special among Spinicaudata due to its very fast development and short life-cycle, which enables them to survive in short-lived habitats and by which they might escape competition and predation from slower developing taxa, as well as the presence of hermaphrodites instead of females (Bellec and page 16 of 23 Zoological Studies 59:44 (2020) Rabet 2016). The latter enables the colonization of new habitats from single resting eggs, which greatly improves dispersal effectiveness.

The Australian fauna is noteworthy not only because of its exceptional diversity (Schwentner et al. 2015b), but it apparently evolved independently from other regions even when Australia was still connected to other Gondwanan landmasses. This seems to have been the case for the Limnadopsis-Paralimnadia-Australimnadia clade within Limnadiidae and the Australian Ozestheria clade. In both cases, age estimates suggest that the Australian clades (134 and 94 mya, respectively) evolved long before the final separation of Australia from the rest of Gondwana [START_REF] Beaulieu | A Southern Hemisphere origin for campanulid angiosperms, with traces of the break-up of Gondwana[END_REF], which occurred around 50 mya. In this relatively long timespan, apparently no exchange between Australia and other continental masses occurred. Such patterns of ancestral regionalization were also found in many other old Gondwanan lineages (e.g., [START_REF] Murienne | A living fossil tale of Pangaean biogeography[END_REF]. Australia was linked to the rest of Gondwana primarily via Antarctica, the only continent without extant Spinicaudata but with a rich fossil record of these animals [START_REF] Shen | Jurassic conchostracans from Carapace Nunatak, southern Victoria Land. Antarctica[END_REF]. Antarctica may have restricted exchange between Australia and other regions of Gondwana simply by geographic distance (isolation by distance). The age of the Australian Eocyzicus clade roughly coincides with the final break-off of Australia, implying that Australia was colonized by Eocyzicus around that time. Again, Eulimnadia may be the only taxon that colonized Australia after it separated from Gondwana, potentially as Australia drew closer to Asia. It has been suggested that Eoleptestheria cf. ticinensis invaded Australia recently from China (Timms 2009a); however, our phylogenetic analyses, as well as previous analyses of the genetic diversity within the Australian populations (Schwentner et al. 2015b), suggest a longer presence of this taxon in Australia. Notably, for the large branchiopod taxa Cyclestherida and Notostraca, younger ages have been assumed for the divergence of Australian and non-Australian species [START_REF] Schwentner | Cyclestheria hislopi (Crustacea: Branchiopoda): a group of morphologically cryptic species with origins in the Cretaceous[END_REF][START_REF] Mathers | Multiple global radiations in tadpole shrimps challenge the concept of 'living fossils[END_REF]. In these taxa, dispersal to or from Australia apparently occurred more recently, probably after Australia and Asia moved closer. The case of the notostracan Triops is particularly interesting as the putatively closest relatives to the Australian fauna occur in North America [START_REF] Mathers | Multiple global radiations in tadpole shrimps challenge the concept of 'living fossils[END_REF].

The age estimates presented herein may also help to improve our understanding of the relationships between fossil and extant taxa [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. We provide the first molecular clock based age estimated for all extant families and many genera. Our results suggest that within each extant family only one to three extant lineages date back more than 150 million years and the main divergence took place within the last 100 million years. Thus the majority of fossil families probably went extinct without any extant representatives; this may be particularly true for the rich Permian and Carboniferous fauna as crowngroup Spinicaudata may have only originated around that time. The similar carapace shapes of Cyzicidae and Eocyzicidae might go back to comparable carapace shapes already known from different Euestheriidae fossils since the Permian [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. Nevertheless, peculiar similarities in carapace shape between fossil and extant taxa may be generally due to convergence rather than evolutionary stasis. For example, the carapace of the fossil Palaeolimnadiopseidae Defretin-Le Franc, 1965 has large similarity to extant species of Limnadopsis and Australimnadia (for example, compare [START_REF] Gallego | First record of the family Palaeolimnadiopseidae Defretin-Le Franc, 1965 (Crustacea-Conchostraca) in the Triassic of Argentina[END_REF][START_REF] Gallego | Conchostracos (Crustaceae-Conchostraca) paleozoicos de la Región de Antofagasta, norte de Chile[END_REF][START_REF] Gallego | First record of the family Palaeolimnadiopseidae Defretin-Le Franc, 1965 (Crustacea-Conchostraca) in the Triassic of Argentina[END_REF] with Timms 2009b or Timms and Schwentner 2012) and it has been suggested that Palaeolimnadiopseidae are the ancestors of Limnadopsis [START_REF] Zhang | Fossil Conchostraca of China[END_REF], but questioned by [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. Palaeolimnadiopseidae date back as far as the Upper Permian (summarized in Gallego 2005) long before the inferred evolution of Limnadiidae, potentially even before the evolution of crown-group Spinicaudata. Of course, it is possible that some younger species that have been assigned to Palaeolimnadiopseidae belong to the stem lineage of extant Limnadiidae and are not related to the older taxa [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF]. Our divergence time estimates may help to improve the current hypotheses of how such fossil and extant taxa may have been related.

Species Diversity of Spinicaudata

Detailed population-based molecular genetic and integrative taxonomic approaches have revealed much higher species diversities for Spinicaudata (e.g., [START_REF] Schwentner | An integrative approach to species delineation incorporating different species concepts: a case study of Limnadopsis (Branchiopoda: Spinicaudata)[END_REF][START_REF] Schwentner | Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure[END_REF]Schwentner et al. 2015a b; b;[START_REF] Weeks | Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)[END_REF]) and other 'large Branchiopoda' like Triops (e.g., [START_REF] Korn | Phylogeny, molecular ecology and taxonomy of southern Iberian lineages of Triops mauritanicus (Crustacea: Notostraca)[END_REF][START_REF] Mathers | Multiple global radiations in tadpole shrimps challenge the concept of 'living fossils[END_REF][START_REF] Meusel | Molecular and morphological delimitation of Australian Triops species (Crustacea: Branchiopoda: Notostraca) -large diversity and little morphological differentiation[END_REF][START_REF] Vanschoenwinkel | Toward a global phylogeny of the "living fossil" crustacean order of the Notostraca[END_REF]. Several species that were assumed to be morphologically variable could be shown to represent an amalgam of multiple, morphologically differentiated, species (e.g., [START_REF] Korn | Phylogeny, molecular ecology and taxonomy of southern Iberian lineages of Triops mauritanicus (Crustacea: Notostraca)[END_REF]Schwentner et al. 2012b;[START_REF] Meusel | Molecular and morphological delimitation of Australian Triops species (Crustacea: Branchiopoda: Notostraca) -large diversity and little morphological differentiation[END_REF][START_REF] Tippelt | Taxonomic assessment of Australian Eocyzicus species (Crustacea: Branchiopoda: Spinicaudata)[END_REF]. However, extensive overlaps of intraspecific variability and interspecific variation are prevalent also in these species and their initial delimitation would have been difficult based solely on morphological characters. The majority of these studies have been conducted on the Australian fauna, which now appears to be the continent with the highest extant clam shrimp diversity, harbouring roughly one third of all spinicaudatan page 17 of 23 Zoological Studies 59:44 (2020) species (Schwentner et al. 2015b). However, the spinicaudatan fauna of other continents have not been studied as extensively. In the analyses presented herein, many species were represented by single individuals or were studied from a few populations only. Despite this relatively sparse intraspecific sampling, instances of putatively cryptic species were revealed in Africa, North America and Europe; in the case of Leptestheria rubidgei even within a single population. On the one hand, this suggests that the species diversity of Spinicaudata may be underestimated on local and global scales, on the other hand it shows that the species level taxonomy of many spinicaudata taxa requires revision. Taxonomic revisions that combine detailed morphological and molecular genetic data will become indispensable to assess the true diversity of Spinicaudata and will probably reveal many more currently cryptic species.

to the one obtained by [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF]. This analysis was performed to obtain node age estimates within Spinicaudata for the subsequent molecular clock analyses using the four gene data set. (download) Eocyzicus, thus also no prior was defined for their divergence (Fig. S13 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 [START_REF] Wolfe | Fossil calibrations for the arthropod Tree of Life[END_REF]) and Limnadiidae + Eocyzicus + Leptestheriidae (minimum age 380 mya based on the assumption that Vertexioidea (known since the mid Devonian) is ancestral to Limnadiidae, while Eosestherioidea is ancestral to Leptestheriidae and Cyzicidae following [START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF], respectively (Fig. 4 S1. List of all individuals studied. For each individual, we provide species names, the species name under which the respective genetic resources have been deposited in GenBank (only applicable for sequences obtained from earlier studies), details on the collection locality and collection event (for sequences obtained from GenBank only the country is provided), collection or specimen numbers used to identify the specimens, the respective citations for published sequences and the GenBank accession numbers for the four genes.

Fig. 2 .

 2 Fig. 2. Phylogenetic relationships of Spinicaudata based on COI, 16S rRNA, EF1α and 28S rRNA inferred with MrBayes. Only individuals with at least two of the four loci available were included and 3rd codon positions of COI were excluded (Matrix 2). For each individual, the country of origin and the collection or voucher number are provided (TableS1), if no collection or voucher number was available one of the GenBank numbers is provided. 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.

  page 8 of 23 Zoological Studies 59:44 (2020)

Fig. 3 .

 3 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.,[START_REF] Rogers | A new Eocyzicus (Branchiopoda: Spinicaudata) from Taiwan, with a review of the genus[END_REF]. 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.

  page 11 of 23 Zoological Studies 59:44 (2020) genus Eulimnadia (Figs. 2, 4, Figs. S1-S3, S12-S14). 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-S3, S12-S14). 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

Fig. 4 .

 4 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.

  Daday, 1914: 193 sensu Rogers et al. 2017

Fig. S12 .

 S12 Fig. S12. Constrained molecular clock dated phylogeny employing minimum age 255 mya for Limnadiidae + Eocyzicus + Leptestheriidae. 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. The topology was constrained to enforce a sister group relationship of Leptestheriidae and Eocyzicus (see Fig. 4 for the unconstrained 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 and 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, (D) 64.8 mya with a sigma of 32 and (E) 146 mya with a sigma of 69. Branches are color-coded according the geographic origin of the specimens. Posterior probabilities are provided for each branch. (download) Fig. S13. Unconstrained molecular clock dated phylogeny employing minimum age 380 mya for Limnadiidae + Eocyzicus + Leptestheriidae. 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.The topology was not constrained to enforce a sister group relationship of Leptestheriidae and Eocyzicus, thus also no prior was defined for their divergence (see Fig.S14for the constrained analysis). Calibration points (6) and (11) are based on fossils for Diplostraca (minimum age 386.9 mya based on Leaia chinensis following[START_REF] Wolfe | Fossil calibrations for the arthropod Tree of Life[END_REF]) and Limnadiidae + Eocyzicus + Leptestheriidae (minimum age 380mya based on the assumption that Vertexioidea (known since the mid Devonian) is ancestral to Limnadiidae, while Eosestherioidea is ancestral to Leptestheriidae and Cyzicidae following[START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF], respectively (Fig.4and Fig. S12 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) (see Figs. S10 and S11) and coded as normal distributed

  Fig. S14. Constrained molecular clock dated phylogeny employing minimum age 380 mya for Limnadiidae + Eocyzicus + Leptestheriidae. 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. The topology was constrained to enforce a sister group relationship of Leptestheriidae andEocyzicus, thus also no prior was defined for their divergence (Fig.S13for 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 380 mya based on the assumption that Vertexioidea (known since the mid Devonian) is ancestral to Limnadiidae, while Eosestherioidea is ancestral to Leptestheriidae and Cyzicidae following[START_REF] Astrop | Phylogenetic relationships between living and fossil spinicaudatan taxa (Branchiopoda Spinicaudata): Reconsidering the evidence[END_REF], respectively (Fig.4and Fig. S12 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 and S11) and coded as normal distributed priors: (A) 403 mya with sigma 10, (B) 155.6 mya with sigma 70, (C) 61.6 mya with sigma 30, (D) 67 mya with sigma 35 and (E) 154 mya with sigma 75. Branches are colorcoded according the geographic origin of the specimens. Posterior probabilities are provided for each branch. (download) TableS1. List of all individuals studied. For each individual, we provide species names, the species name under which the respective genetic resources have been deposited in GenBank (only applicable for sequences obtained from earlier studies), details on the collection locality and collection event (for sequences obtained from GenBank only the country is provided), collection or specimen numbers used to identify the specimens, the respective citations for published sequences and the GenBank accession numbers for the four genes. Collection numbers refer to the Australian Museum Sydney (AM P.), Museum of Comparative Zoology, Harvard University (MCZ IZ) and the Center of Natural History in Hamburg (ZMH K). (download)

  Collection numbers refer to the Australian Museum Sydney (AM P.), Museum of Comparative Zoology, Harvard University (MCZ IZ) and the Center of Natural History in Hamburg (ZMH K). (download) page 23 of 23 Zoological Studies 59:44 (2020)

Table 1 .

 1 List of primers used in this study. For COI and EF1α different combinations of the available primers were used

	Primer name	Marker Sequence 5'-3'

  using the uncorrelated gamma multipliers (-ugam;[START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF]) relaxed clock model with free parameters for birth death priors (-bd) on divergence times. The consensus tree was recovered using the bpcomp command. The calibrated trees were visualized with FigTree 1.4.3.Eleven fossil calibration points were applied in this first molecular clock analysis. For all calibration points, the maximum age was set to 636.1 mya (million years ago) based on the youngest Lagerstätten without known Eumetazoa (following Wolfe et al. 2016). Minimum ages for calibration points were: (1) 497 mya for crown-group Allotriocarida based on Rehbachiella kinnekullensis Muller, 1983 (following Wolfe et al. 2016), representing the root of the tree, (2) 495 mya for the split between Branchiopoda and Remipedia based on the oldest stem group branchiopod (following Harvey et al. 2012), (3) 405 mya for crown-group Branchiopoda based on Lepidocaris rhyniensis (following Olesen 2009 and Wolfe et al. 2016), (4) 405 mya for crowngroup Phyllopoda based on Castracollis wilsonae Fayers & Trewin, 2003 (following Olesen 2009), (5) 121.8 mya for crown-group Notostraca based on Chenops yixianensis Hegna & Dong, 2010 (following Wolfe et al. 2016), (6) 386.9 mya for crown-group Diplostraca based on Leaia chinensis, which was placed by Wolfe et al. (2016) as either crown-group Diplostraca or Onychocaudata (setting this constraint for Diplostraca is more conservative), (7) 250 mya for crown-group Cladoceromorpha based on the oldest known fossil Cyclestherioides (following Raymond 1946 and Negrea et al. 1999), (8) 175 mya for the split between Ctenopoda and all other Cladocera based on fossil ctenopod Smirnovidaphnia smirnovi Kotov, 2007 (following van Damme and Kotov 2016 and Wolfe et al. 2016), (9) 145 mya for the split between Daphnia pulex Leydig, 1860 and its closest relative in the data set (Ceriodaphnia quadrangula (Müller, 1875)) based on the oldest Daphnia fossils (following van Damme and Kotov 2016), (10) 145 may for the split between Simocephalus vetulus (Müller, 1776) and its closest relative in the data set (Scapholeberis mucronata (Müller, 1776)) based on the oldest known Simocephalus fossils (following van Damme and Kotov 2016) and (11) 255 mya for the split between Limnadiidae and Eocyzicus + Leptestheriidae based on the oldest known Perilimnadiidae fossils (following

	Zoological Studies 59:44 (2020)	page 6 of 23

  2014) as has the clade comprising

	Zoological Studies 59:44 (2020)	page 13 of 23

  partim) Diagnosis: Cephalic fornices extending anteriorly to rostral apex. Rostrum variable, blunt to acute, long or short, generally triangular to subquadrate in lateral view. Rostrum with or without an apical spine, usually without. Compound eyes fused medially, sometimes projecting in smoothly arcuate ocular tubercle. Frontal organ sessile. Occipital notch present. Carapace thick, generally rounded. Carapace dorsal margin smooth, lacking carinae, hinge line straight. Carapace with or without pigmentation, growth lines obvious, projecting. Umbone present, projecting well above hinge line. Muscle scar rarely visible. Male first two thoracopods with endopod (= movable finger, sensu Olesen 2007) lacking an apical suctorial organ, if modified setae or spines are present these are never with a lateral fringe. Telson without a ventroposterior, posteriorly directed spiniform projection. Eggs 110-170 μm in diameter, spherical and generally lacking ornamentation.

  pro partim Diagnosis: Populations composed of males and females (except C. gynecius which is only composed of hermaphrodites); amplexus is venter to venter. Rostrum subtriangular (usually females) to subquadrate (usually males), depending on age and gender. Angle between rostrum and frons 160° to 180°. Occipital notch either deep and narrow, often closed or very shallow, or absent. Occipital condyle either conical, subacute, length subequal to basal width or low, rounded, length half or less basal width. Rostral spine generally absent (present in C. australis). Carapace valve length ~1.3x

Zoological Studies 59:44 (2020) © 2020 Academia Sinica, Taiwan

Acknowledgments: This work (urn:lsid:zoobank. org:pub:665CB83B-7A2A-445F-9154-B18BDCC A573C) and Eocyzidae fam. nov. (urn:lsid:zoobank. org:act:CF3B4318-483E-47B8-A391-DA6A9E77400F) have been registered with ZooBank. We are greatly indebted to Brian V. Timms, who has made this research possible through his relentless efforts in studying Australian Branchiopoda and has taken MS, NR, SR, and DCR on numerous collecting expeditions across Australia. We thank Mikhail Son (Institute of Marine Biology, Odessa, Ukraine) and Federico Marrone (Università degli Studi di Palermo, Palermo, Italy) for providing specimens as well as two anonymous reviewers for their helpful comments and Thomas Hegna for his patience and support. We thank also Daniela Lupi, Eric Gallerne, Eric Quéinnec, Marco Pagni, Michaël Manuel, Pascal Lluch, Sébastien Lacau for the help in the field or soil sampling. Sequencing costs were financed via internal funds from the Museum of Comparative Zoology and the Faculty of Arts and Sciences to G.G. and from the Center of Natural History (Universität Hamburg) to M.S. and via Emergence project of UPMC and ATM gave to N.R. This work benefited from equipment and services from the iGenSeq core facility, at ICM, Sorbonne-Unversité and SSM in MNHN (Paris).

Availability of data and materials: All sequences were submitted to GenBank (accession numbers MN553596-MN553672 and MN584937-MN585093). Specimens are stored in museum collections (Table S1). Further data can be obtained form the corresponding author upon request.

Consent for publication: All authors consent to the publication of this manuscript.

Authors' contributions: M.S. and D.C.R. conceived and planned the study. M.S. performed all Sanger sequencing and phylogenetic analyses. D.C.R. updated the diagnoses of the taxa. M.S., D.C.R. and N.R. drafted the manuscript. All authors contributed to the final version of the manuscript.

Competing interests:

The authors declare that they have no conflict of interests.

Ethics approval consent to participate: Not applicable. [START_REF] Schwentner | Tetraconatan phylogeny with special focus on Malacos t raca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics[END_REF]. This analysis was performed to obtain node age estimates within Spinicaudata for the subsequent molecular clock analyses using the four gene data set. (download)

Supplementary Materials