New lineages and old species: Lineage diversity and regional distribution of Moina (Crustacea: Cladocera) in China

Accepted Manuscript New lineages and old species: lineage diversity and regional distribution of Moina (Crustacea: Cladocera) in China Yijun Ni, Xiaolin Ma, Wei Hu, David Blair, Mingbo Yin PII: DOI: Reference:

S1055-7903(18)30710-3 https://doi.org/10.1016/j.ympev.2019.02.007 YMPEV 6421

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

13 November 2018 8 February 2019 8 February 2019

Please cite this article as: Ni, Y., Ma, X., Hu, W., Blair, D., Yin, M., New lineages and old species: lineage diversity and regional distribution of Moina (Crustacea: Cladocera) in China, Molecular Phylogenetics and Evolution (2019), doi: https://doi.org/10.1016/j.ympev.2019.02.007

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New lineages and old species: lineage diversity and regional distribution of Moina (Crustacea: Cladocera) in China Yijun Ni1, Xiaolin Ma1, Wei Hu1, David Blair2 and Mingbo Yin1* 1

MOE Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life

Science, Fudan University, Songhu Road 2005, Shanghai, China 2

College of Marine and Environmental Sciences, James Cook University, Townsville Qld 4811,

Australia.

*

Corresponding author:

Mingbo Yin; [email protected]

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Abstract: The distribution and genetic diversity of freshwater zooplankton is understudied in the Eastern Palearctic. Here, we explored the lineage diversity and regional distribution of the genus Moina in China. Members of this genus are often keystone components of freshwater ecosystems and have been frequently subjected to toxicological and physiological studies. Four species of Moina were identified, based on morphology, in 50 of 113 Chinese water bodies examined, and their phylogenetic position was analyzed using both a mitochondrial (mitochondrial cytochrome c oxidase subunit I; COI) and a nuclear marker (the nuclear internal transcribed spacer; ITS-1). Both molecular markers identified four clades corresponding broadly to the morphological species. Mitochondrial DNA analysis showed the presence of four species complexes with eleven lineages across China, five of which were new. However, some lineages (and even individual haplotypes) were widespread in Eurasia, suggesting an ability to disperse over long distances. In contrast, a few lineages exhibited restricted distributions. The nuclear phylogeny also recognized four species of Moina within China and seven very distinct clades. Interestingly, one specimen possessing Moina cf. micrura mtDNA had ITS-1 alleles of the M. cf. brachiata clade. This discordance between mtDNA and nuclear ITS-1 phylogenies is indicative of interspecific introgression and hybridization. Additionally, our COI phylogeny showed apparent paraphyly in two Moina species groups, suggesting introgression of their mitochondrial genomes. Our data shows the regional distribution/diversity of the Moina species complex in a Eurasian context.

Keywords: Moina; genetic lineages; COI gene; ITS-1; gene introgression; Eurasia

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Introduction: Until recently, there was a widely accepted view that species with large population size and strong dispersal abilities, especially freshwater invertebrate species, are cosmopolitan in their geographic distributions (Baas-Becking, 1934). However, extensive genetic studies have revealed that many “species” originally assumed to be widespread are in fact mosaics of several cryptic species (e.g. Andrews et al., 2014; Bickford et al., 2007; Darling et al., 2007; Feulner et al., 2006; Hebert et al., 2004; Penton et al., 2004). This has often been observed in zooplankton, such as copepods and cladocerans (e.g. Cornils et al., 2017; Petrusek et al., 2012). For example, morphologically identical but genetically paraphyletic taxa have been observed in the copepod Hemidiaptomus ingens s.l. (Marrone et al., 2013) and the cladoceran Daphnia pulex (Colbourne et al., 1998).

The genus Moina Baird (Cladocera: Moinidae) is a speciose genus of anomopods, close relatives of the Daphniidae (Padhye and Dumont, 2014). They are important components of freshwater ecosystems, as grazers of phytoplankton and as key prey for fish larvae (Lampert and Sommer, 1999). Although Moina species have been frequently subjected to toxicological and physiological studies (e.g. Jia et al., 2018; Kwak et al., 2018; Mangas-Ramirez et al., 2002), this genus has received less attention with respect to systematics (e.g. Bekker et al., 2016; Hudec, 1990; Kotov et al., 2005; Mirabdullaev, 1993; Mirabdullayev, 1998; Padhye and Dumont, 2014). The first study using genetic markers showed that populations of M. macrocopa from the Czech Republic and Uganda were indeed conspecific, whereas “M. micrura” from Europe and Australia belonged to two different biological species based on results of experimental crosses (Petrusek et

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al., 2004). Later, a DNA-barcoding study explored variation within M. brachiata in Hungary and identified several highly divergent lineages (Nedli et al., 2014). More recently, a comprehensive study showed a high genetic diversity in the northern Eurasia Moina species using the mitochondrial COI gene (Bekker et al., 2016). For the remainder of this paper, taxon names used will be those of Bekker et al. (2016), unless otherwise stated. There are currently more than 25 named species of moinids (Kotov et al., 2013) and cryptic species/genetic lineages undoubtedly remain to be discovered (Padhye and Dumont, 2014). However, there have been no notable taxonomic studies on Moina in China, and no published molecular studies.

Similar to other water fleas, Moina utilizes cyclical parthenogenesis, in which one or more generations of parthenogenetically produced females alternate with a sexual generation with males producing sperm and females producing haploid eggs (Grosvenor and Smith, 1913): fertilized eggs then enter diapause. When the environmental conditions are ideal, parthenogenesis can lead to rapid population growth. Only when unfavourable conditions arise, such as food shortage, overcrowding, or changes in temperature, do Moina individuals switch to sexual reproduction and produce dormant eggs (Dabramo, 1980). Birds are believed to be the key vectors for the passive dispersal of dormant eggs of freshwater zooplankton (Havel and Shurin, 2004), and it seems likely that they could introduce dormant Moina eggs across Eurasia.

The present study aims at an assessment of the genus Moina in China, using sequences of two gene fragments: the mitochondrial cytochrome c oxidase subunit I (COI), and the nuclear internal transcribed spacer (ITS-1). We analysed specimens from China and also utilized

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previously published sequences from Eurasia (Bekker et al., 2016). Using two independent methods of species delimitation, we estimated the existence and number of genetic lineages within Moina and placed these within a framework of known species as defined by morphology. Given our use of both a nuclear and a mitochondrial marker, we also considered that our data might provide some evidence of hybridization within Moina. The sexual phase in the life-cycle could result in the formation of interspecific hybrids if closely related species co-occur, mate and produce sexual (ephippial) eggs (Hebert, 1985). Hybridization has been described in cladocerans, for example Simocephalus, Pleuroxus and Bosmina (Schwenk and Spaak, 1995), and is especially common in Daphnia (e.g. Keller et al., 2008; Schwenk and Spaak, 1995; Xu et al., 2013; Yin et al., 2014; Yin et al., 2010). However, no hybrids have ever been reported in Moina, despite the high diversity of species and extensive cryptic species/lineages (Kotov et al., 2013; Padhye and Dumont, 2014). We therefore also used our molecular data in a preliminary test of the hypothesis that hybridization can occur between Moina species, as observed in other zooplankton (Hebert, 1985).

Materials and methods Terms used 1, “morphospecies” or “morphological species” are distinguished from others by their morphology. In our study, names were assigned to morphospecies by comparing their COI sequences with those of named species/ species complexes in GenBank. 2, A lineage (or genetic lineage) is defined here as a strongly supported (usually sub-specific) grouping of sequences with, frequently, short internal branches and low support for internal

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nodes. In this paper, a lineage is also a group of sequences recognized as distinct by the speciesdelimitation programs, bPTP and GMYC (see below). 3, “species complex” indicates a group of closely related species that are very similar in appearance to the point that the boundaries between them are often unclear. 4, “cryptic species” are apparently morphologically identical to each other but belong to different species based on genetic analysis – cryptic species are generally grouped within a species complex.

Sampling and morphological characterization Moina specimens were recovered from 50 of 113 lakes sampled across China. Fifteen of the water-bodies were located in the Eastern Plain, 2 in the Inner Mongolia-Xinjiang Plateau, 15 in the Qinghai-Tibet Plateau, 1 in the Yunnan-Guizhou Plateau and the remaining 17 in the Northeast Plain (Figure 1 and Table 1). Samples were collected with a 125-μm plankton net hauled vertically through the entire water column at two or three different sites per lake. Samples from different locations in the same lake were pooled and preserved in 95% ethanol. Prior to DNA extractions, all specimens were placed into a drop of water on a slide and were identified morphologically (Chiang and Du, 1979; Goulden, 1968) and photographed under a stereomicroscope. The morphological characters of adult female Moina, including body length (defined as the length between the upper edge of the head and the lower edge of the postabdomen), body transparency and form of head and body (Goulden, 1968), were recorded to distinguish the taxa. The body length of 20 adult individuals per species were measured under the stereomicroscope. The differences in the body length among four species were calculated by one-way ANOVA in SPSS 24.

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Molecular analyses Individuals from 50 populations were processed for molecular analyses (Table 1). DNA was extracted by H3 buffer with proteinase K (60 μL), containing 10 mM Tris-HCl, 0.05 M KCl, 0.005% Tween 20, 0.005% NP-40 and 10 mg/ml proteinase K (MERCK, Germany). Samples were incubated for 10-16 hours at 55 °C in a water-bath with mild shaking. Afterwards, the proteinase K was irreversibly denatured by a 12 min incubation at 95 °C, the tube centrifuged briefly and stored at 4 °C before use. We amplified a 680-bp segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene using a standard primer pair LCO1490 and HCO2918 (Folmer et al., 1994). The PCR was carried out in a total volume of 20 μL, consisting of 1 × PCR buffer (10 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 50 mM KCl), 2.5 mM of each dNTP, 0.5 μM of each primer, 2 units of Taq DNA polymerase (SuperTherm DNA polymerase, Taq HS from TAKARA BIO INC., California, USA) and 2 μL of genomic DNA. The PCR temperature profile used for the COI amplification was as follows: incubation at 94 °C for 1 min, then 40 cycles of 1 min at 94 °C, 1.5 min at 40 °C and 1.5 min at 72 °C; this was followed by a final incubation for 6 min at 72 °C. PCR products of the COI locus were purified and sequenced in the forward direction on an ABI PRISM 3730 DNA capillary sequencer by Invitrogen Trading Co., Ltd (China). A total of 508 individuals were sequenced at the COI locus, and then 1-13 individuals from each COI lineage (50 individuals in total; Table 1) were chosen for sequencing of the nuclear internal transcribed spacer (ITS-1). A 700-bp segment of ITS-1 was amplified using primers 18SD and 5.8BR (Taylor et al., 2005). The PCR mixture was the same as for COI (except for the primers used). The ITS-1 amplification cycling conditions were as follows: incubation at 94 °C for 1 min, then 10 cycles of 1 min at 94 °C, 1 min at 53 °C and 2 min at

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72 °C, 30 cycles of 1 min at 92 °C, 2 min at 53 °C and 1 min at 72 °C; this was followed by a final incubation for 7 min at 72 °C. Because ITS-1 fragments sometimes contained multiple heterozygous sites, cloning was performed to obtain unambiguous chromatograms: the PCR products were recovered from an agarose gel and inserted into a pMD®19-T Vector plasmid (TaKaRa, Beijing, China) using pGEM®-T Vector System I Kit (Promega, Beijing, China) and transformed into E. coli DH5α (TsingKe Biotech Co., Ltd, Beijing, China). Recombinant clones harboring the insert were verified by colony PCR (cycling conditions as above). Plasmids were then extracted from bacteria using HiPure Plasmid Micro Kit (Magen, Guangzhou, China) according to the manufacturer's protocol. Up to 15 clones were sequenced for each PCR product from ITS-1: only identical sequences obtained at least twice per PCR product were chosen for further analysis. All ITS-1 PCR products were sequenced using a forward primer on an ABI PRISM 3730 DNA capillary sequencer by Invitrogen Trading Co., Ltd (China). All the chromatograms were carefully checked and manually corrected for scoring errors in MEGA 6 (Tamura et al., 2013), and the quality scroes of the sequences were examined with Chromas Lite Version 2.1 (Technelysium Pty. Ltd., South Brisbane, Australia). Chromatograms with double peaks or noise were resequenced in the reverse direction, and only chromatograms of high quality (Phred quality scores > 40) were used for the genetic analysis. All newly obtained sequences were submitted to GenBank under accession numbers: COI: MK394724-MK394776 and ITS-1: MK394777-MK394799.

Sequence alignment and genetic diversity For COI, unique haplotypes were identified in DNASP 5.10 (Librado and Rozas, 2009) and MUSCLE (Edgar, 2004) implemented in MEGA 6 was used to align the sequences which were

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subsequently translated into amino acids in order to examine the presence of stop codons that would indicate potential pseudogenes. All haplotypes were then aligned together with 308 reference sequences retrieved from GenBank (Tables S1 and S2), using the Clustal W algorithm (Thompson et al., 1994) in MEGA 6. One hundred and fifty-six out of 308 reference sequences, which had detailed location information (Bekker et al., 2016), were from 50 Moina populations across nine regions, including the Central Siberian Plateau (4 populations), the East European Plain (18 populations), East Siberia (10 populations) and the Western Siberian Plain (7 populations) of Russia, Hungary (1 population), Kazakhstan (4 populations), Mongolia (4 populations), Ukraine (1 population) and Tibet of China (1 population), see Table S1. The remaining 152 reference sequences, for which geographical information was not available, were only included in phylogenetic analyses, see Table S2. For ITS-1, unique haplotypes were detected in DNASP 5.10, and then aligned using Clustal W algorithm in MEGA 6. No reference sequences of Moina ITS-1 were available from GenBank or other public databases. For each morphospecies (consistent with the identification based on COI phylogeny), the number of haplotypes (N2), haplotype (H) and nucleotide diversity (π) were calculated in DNASP 5.1 (Librado and Rozas, 2009) for both COI and ITS-1 markers. Intra-individual differences between ITS-1 alleles in heterozygotes was calculated in MEGA 6.

Phylogenetic analyses and divergence time estimation Potential loss of phylogenetic signal resulting from substitution saturation at the COI was inspected using the test of Xia et al. (2003) implemented in DAMBE 5 (Xia, 2013). Then, a phylogenetic tree was constructed for the COI marker using the Bayesian method in BEAST 2 (Bouckaert et al., 2014), with every 1000 generations recorded among 10,000,000, a burn-in of

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25%, and the final 10,000 trees summarized using TreeAnnotator. GTR+G+I was estimated to be the best-fit substitution model by the corrected Akaike Information Criterion in jModeltest v. 2.1.7 (Darriba et al., 2012). A strict molecular clock was applied and all other tree priors were left at their default values. Tracer v1.6 (Rambaut et al., 2018) was used to ensure that enough generations were computed. Ceriodaphnia, a member of the Cladocera phylogenetically close to Moina, was used as an outgroup. A fossil-based calibration point for the most recent common ancestor of Ceriodaphnia sp. and Moina was set at 145 mya and assigned a 10% standard deviation. This calibration point was based on fossil evidence for Daphnia + Simocephalus (both amonopod Cladocera) (Kotov and Taylor, 2011).

Detection of genetic lineages and phylogeographic analyses To test the hypothesis that Moina is a complex of reproductively isolated species/lineages, two independent species delimitation methods were applied to the sequences of both markers (COI and ITS-1): the general mixed Yule coalescent model (GMYC, Pons et al., 2006) and Poisson tree processes methods (PTP, Zhang et al., 2013). The GMYC is a likelihood-based method for delimiting species by fitting within- and between-species branching models to reconstruct gene trees, using an ultrametric tree. The GMYC modeling was performed using the “splits” package (Ezard et al., 2009) in R 2.15 (R Development Core Team, 2009). The PTP calculations were conducted on the bPTP websever (http://species.h-its.org/ptp/), with 100,000 MCMC generations, thinning set to 100 and burnin at 25% and performing a Bayesian search. The input phylogenetic tree was generated with BEAST 2 (see above). To visualize genealogical relationships among Moina lineages, a network of COI haplotypes (including 156 reference sequences from GenBank; Table S1) was constructed using HAPLOVIEWER (Salzburger et al., 2011). The maximum 10

likelihood tree inferred with MEGA 6 with the best model (GTR+G+I; by jModeltest v. 2.1.7) was used as input. Additionally, the maximum likelihood (ML) analysis (best model GTR+G+I; by jModeltest v. 2.1.7) was performed with MEGA 6 and bootstrap resampled 1000 times, for phylogroups of Moina, M. cf. brachiata and M. cf. macrocopa, respectively. All 308 reference sequences (see Tables S1 and S2) from GenBank were included in this analysis. Clades were collapsed using FigTree (http://tree.bio.ed.ac.uk/software/figtree/) with the collapse module. Uncorrected p-distances between Moina lineages were calculated in MEGA 6 based on COI. To estimate genetic differentiation among Moina populations (per species), the fixation index FST was calculated in Arlequin 3.11 with 104 permutations. Finally, the correlation between pairwise geographical distance and pairwise FST was computed using a Mantel test (104 permutations, in the Isolation by Distance Web Service, version 3.15, (Jensen et al., 2005)). Moina cf. salina was excluded from the analysis as only three populations (and the sample size of XDP is 1) from this species were detected in this study.

Results Morphology We identified four morphospecies of Moina in China. The names used by Kotov and collaborators (Bekker et al., 2016) for Moina taxa have been followed. The four species differ in morphological characteristics: M. cf. brachiata is caesious and opaque whereas the other three are pale and hyaline; M. cf. brachiata, M. cf. micrura and M. cf. salina have a supraocular depression on the head, their dorsum of valves is elevated behind the head when embryos are present, and their posterior part is convex; but this is not the case for M. cf. macrocopa (Figure S1). Additionally, M. cf. macrocopa has the largest body length (range:1071.95 -1545.99 μm; 11

mean = 1298.80 μm; stdev = 120.00 μm), followed by M. cf. brachiata (range: 964.43-1423.23 μm; mean = 1147.79 μm; stdev = 109.81 μm) and M. cf. salina (range: 789.24- 1072.38 μm; mean = 882.73 μm; stdev = 75.42μm), and M. cf. micrura is smallest (range: 520.97- 702.05 μm; mean = 641.42 μm; stdev = 37.84 μm), see Figure S2.

Genetic diversity Sequences were successfully obtained from 508 Moina individuals at the COI locus (478 bp in the aligned dataset) and 50 individuals at ITS-1 (27 heterozygotes and 23 homozygotes, resulting in a total of 77 sequences; 636 bp in the aligned dataset). Fifty-three unique COI haplotypes and 23 unique ITS-1 alleles were detected. None of the COI sequences exhibited characteristics of nuclear pseudogenes (frame shifts or premature stop codons). For each species, the haplotype diversity (H) of COI ranged from 0 to 1 (mean = 0.378), and the nucleotide diversity (π) ranged from 0 to 0.082 (mean = 0.014; Table 1). The haplotype diversity (H) of ITS-1 ranged from 0.582 to 1 (mean = 0.828), and the nucleotide diversity (π) ranged from 0.001 to 0.111 (mean = 0.046). The amount of intra-individual difference between alleles of heterozygotes at ITS-1 ranged from 1 bp to 13 bp (data not shown).

Phylogeny and divergence time estimation Two independent species-delimitation methods (i.e. GMYC and bPTP) based on the COI Bayesian tree identified 28 and 29 Moina mitochondrial lineages, respectively. Only a difference between the two methods was detected in recognition of one or two lineages in a small group of M. cf. macrocopa haplotypes. Both species-delimitation methods identified eleven lineages in

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China (Figure 2) that were well-supported with posterior probability (PP) values > 0.85. The genetic lineages observed in China were grouped in four clades that corresponded for the most part to the four morphologically described species. Of Moina species for which relevant sequence data were available, only M. lipini was not represented in China. Among the eleven Chinese lineages, four (B, ⅰ, ⅱ and G) belonged to M. cf. brachiata, four (ⅲ, ⅳ, ⅴ and I) belonged to M. cf. micrura, one (ⅵ) belonged to M. cf. macrocopa, and two (N and O) belonged to M. cf. salina (Figure 2). Five of the eleven lineages in China were new, two (ⅰ and ⅱ) belonged to M. cf. brachiata and three (ⅲ, ⅳ and ⅴ) belonged to M. cf. micrura (Figure 2 and S3). Our COI phylogeny also suggests that M. cf. micrura and M. cf. macrocopa, as currently defined, are paraphyletic groups. One lineage of M. cf. micrura is more closely related to the M. cf. macrocopa clade than to the remaining M. cf. micrura lineages. Similarly, M. cf. macrocopa is paraphyletic with respect to M. lipini. The most recent common ancestor of all included Moina lineages on the basis of the COI alignment was estimated to be around 117.3 Mya (95% HPD: 85.2-150.7 mya). Moina cf. brachiata and M. cf. micrura diverged around 90.6 Mya (95% HPD: 65.3-119.2 mya); M. lipini and M. cf. macrocopa diverged around 58.3 Mya (95% HPD: 36.485.3 mya) (Figure 3). The genetic distances (uncorrected p-distances) based on COI ranged from 0.025 to 0.204 between lineages (data not shown).

Geographical distribution of Moina lineages Moina cf. brachiata was the most widely distributed taxon in Eurasia where it has been detected in ten out of thirteen sampled regions, except for Hungary, and was also not found on the Eastern Plain and the Yunnan-Guizhou Plateau of China (Figure 1). Two new lineages from this species

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were abundant but restricted to the Qinghai-Tibet Plateau in China (Figure 4). Moina cf. micrura was the second most widely distributed species in Eurasia, but it was the most widely distributed species in China where it was found in four regions but not in the Qinghai-Tibet Plateau. Three new lineages of M. cf. micrura were detected in China: one in the Eastern Plain and the Northeast Plain and other two were restricted to the Eastern Plain (Figure 4). Only one M. cf. macrocopa lineage (ⅵ), which was widely distributed in the Eastern Plain and the Qinghai-Tibet Plateau, was detected in China. This lineage was also present in the Central Siberian Plateau, East Siberia and the Western Siberian Plain of Russia (Figure 4). Moina cf. salina was rare and detected from three Chinese lakes (two adjacent ponds in the Northeast Plain and one from the Qinghai-Tibet Plateau; Figure 1). Different Moina species co-existed in the same lake across China (Figure 1 and Table 1). For example, M. cf. brachiata and M. cf. macrocopa co-existed in three lakes from the Qinghai-Tibet Plateau (M1C, R3F and SZP), and M. cf. brachiata and M. cf. micrura coexisted in three lakes on the Northeast Plain (KLP, LHH and XHP). Three species (i.e. M. cf. brachiata, M. cf. micrura and M. cf. salina) co-existed in a single lake on the Northeast Plain of China (XDP) (Figure 1 and Table 1). Pairwise FST values ranged from 0 to 1 among M. cf. brachiata populations (Figure 5A), from 0 to 1 among M. cf. micrura populations (Figure 5B) and from 0 to 1 among M. cf. macrocopa populations (Figure 5C). There was evidence of genetic isolation-by-distance for all three tested Moina species, i.e. M. cf. brachiata, M. cf. micrura and M. cf. macrocopa (M. cf. brachiata: R2 = 0.1646, P < 0.001; M. cf. micrura: R2 = 0.04302, P < 0.001; M. cf. macrocopa: R2 = 0.6533, P < 0.001; Figure 5).

Gene introgression

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The two independent species-delimitation methods (i.e. GMYC and bPTP) based on the ITS-1 Bayesian tree (Figure 6) both indicated that the Chinese Moina populations consisted of four species with seven very distinct clades. Interestingly, one specimen possessing M. cf. micrura mtDNA had ITS-1 alleles of the M. cf. brachiata clade, and another specimen possessing M. cf. brachiata mtDNA of lineage G had ITS-1 alleles of lineage B (Table 2 and Figure 6).

Discussion Lineage diversity in Moina from Eurasia In this study, we detected five new mitochondrial lineages across China: two within M. cf. brachiata and other three within M. cf. micrura. Similar lineage diversity within the genus Moina has already been reported in the Czech Republic and Australia (Petrusek et al., 2004), Hungary (Nedli et al., 2014) and Russia (Bekker et al., 2016). Another lineage, ⅵ, which was verified in this study by two independent species-delimitation methods, was previously assigned to lineage M (Bekker et al., 2016). In agreement with the high lineage diversity of Moina from the northern half of Eurasia (Bekker et al., 2016), here, we showed extensive lineage diversity within Moina across China. Some of these identified lineages might in the future be regarded as cryptic species following a distinct evolutionary trajectory (Pfenninger and Schwenk, 2007). Indeed, by using genetic tools, cryptic species have frequently been observed in cladocerans (e.g. Adamowicz et al., 2009; Forro et al., 2008; Petrusek et al., 2012). However, taxonomic recognition of cryptic species in Moina requires further research, for example in-depth morphological analyses. Consequently, we confidently assume that the number of moinid species

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is comparable to that in Daphnia and thus Moina is one of the largest genera of the Cladocera, as proposed by Bekker et al. (2016).

Phylogeography and dispersal Our data suggested that Moina species diverged long ago. This is consistent with findings for other cladocerans: fossil evidence indicates that the main subgenera of Daphnia were already established by 145 Mya (Kotov and Taylor, 2011). These divergences pre-date events such as the Himalayan orogeny. Indeed, the world climate and positions of the main landmasses in the Mesozoic were very different from today, showing the ability of these taxa to persist through geological time and global change. The mtDNA-based phylogeny indicates the common presence of M. cf. brachiata and M. cf. micrura in China, each represented by multiple lineages. These findings are in line with those of Bekker et al. (2016) from the northern half of Eurasia, also based on COI sequences. In that study, five Moina species, together with several unidentified lineages, were detected. Many Moina lineages (and haplotypes) are shared among regions in Eurasia. Specifically, a single haplotype (MSI5) occurs across Eurasia (from China and South Korea in the east, to the East European Plain, Kazakhstan and Hungary), suggesting relatively recent dispersal across long distances. There was a significant association between genetic distance and geographical distance for all three tested Moina species. This indicates lack of panmixia and that there might be potential for regional divergence. However, the scatter plots in Figure 5, especially that for M. cf. micrura indicates a huge range of FST values between pairs of populations separated by any geographical distance. This might reflect occasional dispersal events.

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Although some Moina lineages are widely present in China, five new lineages were found, all showing a smaller, regional distribution instead of a nation-wide distribution. Specifically, lineages ⅰ and ⅱ of M. cf. brachiata were restricted to the Qinghai-Tibet Plateau, whereas lineages ⅲ, ⅳ and ⅴ of M. cf. micrura were only detected in the Eastern Plain/Northeast Plain. Our study strongly shows that it is a common phenomenon for zooplankton taxa, whose distributions were initially assumed to be global, to consist of many lineages, each restricted to a certain region (e.g. Andrews et al., 2014; Colbourne et al., 1998; Cornils et al., 2017).

Gene introgression among Moina lineages Discordance between mtDNA and nuclear ITS-1 phylogenies is indicative of interspecific introgression and hybridization among species/lineages. Here, an individual with M. cf. micrura mtDNA was found unexpectedly in a lineage of the M. cf. brachiata ITS-1 clade. Moreover, another specimen possessing M. cf. brachiata mtDNA of lineage G had ITS-1 alleles which belonged to the M. cf. brachiata mtDNA of lineage B. Mito-nuclear discordances are frequently reported: Toews and Brelsford (2012) reviewed 126 such cases in animals and additional examples have been observed in Cladocera: Daphnia (e.g. Thielsch et al., 2017; Yin et al., 2018) and Diaphanosoma (Liu et al., 2018). Cyto-nuclear discordance most likely results from hybridization and subsequent introgression of the mitochondrial genome (Gompert et al., 2008; Linnen and Farrell, 2007). Indeed, it is especially common in the Daphnia longispina species complex (e.g. Keller et al., 2008; Rellstab et al., 2011; Thielsch et al., 2017; Yin et al., 2014). However, other explanations for mito-nuclear discordance, such as incomplete lineage sorting of

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ancestral polymorphisms (Franco et al., 2015; Mckay and Zink, 2010), or selection acting on mitochondrial genes (Cheviron and Brumfield, 2009; Pavlova et al., 2013), could not be ruled out from this study because no relevant experiment has been reported in the Moina system. Our mtDNA data indicate paraphyly in the genus Moina. However, this could also reflect introgression of mitochondrial genomes of one species into the nuclear background of another as a consequence of hybridization and subsequent back-crossing with one parental species (Funk and Omland, 2003). Paraphyly in molecular trees has already been observed in other freshwater zooplankton taxa, for example, in the cladoceran Daphnia pulex species complex (e.g. Colbourne et al., 1998; Hebert et al., 1989). In the D. pulex complex, many populations of D. pulicaria in North America contain mtDNA genomes derived through introgression with D. pulex, rendering the former paraphyletic with respect to the latter (Hebert et al., 1989).

The internal transcribed spacer was our only nuclear marker. Although, in conjunction with mitochondrial data the ITS-1 can indicate introgression, additional nuclear markers (such as microsatellites) will provide support for introgression/hybridization in China. By applying a set of microsatellite markers, a recent study from China showed that Daphnia hybrids occupy intermediate or occasionally extreme environments whereas their parental species are geographically and ecologically separated (Ma et al., In press). Frequent hybridization, as assumed by Hebert (1985), might be detected in cladocerans if high-resolution genetic markers are applied. Additionally, the samples analyzed in this study were mostly from Eurasia, but Moina has a gobal distribution: much more geographical sampling is required to supplement the published data from Europe and North America in order to place the distribution/diversity of the genus Moina in a gobal context. 18

Conclusions In conclusion, our data revealed extensive genetic diversity of Moina in China and the presence of four distinct species complexes. Different, opposing effects may be influencing populations of Moina across its range. On the one hand, genetic markers demonstrate the capacity for widespread dispersal. Our markers also found some potential region-specific lineages, hinting at the potential for populations to diverge in isolation. Dispersal, sexual processes and hybridization will tend to inhibit the evolution of new species, but to what extent is unclear. We found discordance between mtDNA and nuclear ITS-1 phylogenies, indicative of interspecific introgression and hybridization between Moina species/lineages, a phenomenon that requires further study.

Acknowledgements This research was funded by the National Natural Science Foundation of China (31670380) and Natural Science Foundation of Shanghai (16ZR1402900) to MY. We thank two anonymous reviewers for useful comments on the earlier version of this manuscript.

References: Adamowicz, S.J., Petrusek, A., Colbourne, J.K., Hebert, P.D.N., Witt, J.D.S., 2009. The scale of divergence: A phylogenetic appraisal of intercontinental allopatric speciation in a passively dispersed freshwater zooplankton genus. Mol. Phylogenet. Evol. 50, 423-436. Andrews, K.R., Norton, E.L., Fernandez-Silva, I., Portner, E., Goetze, E., 2014. Multilocus evidence for globally distributed cryptic species and distinct populations across ocean gyres in a mesopelagic copepod. Mol. Ecol. 23, 5462-5479. Baas-Becking, L., 1934. Geobiologie of inleiding tot de milieukunde. W.P. van Stockum and Zoon, The Hague, The Netherlands.

19

Bekker, E.I., Karabanov, D.P., Galimov, Y.R., Kotov, A.A., 2016. DNA barcoding reveals high cryptic diversity in the North Eurasian Moina species (Crustacea: Cladocera). Plos One 11. Bickford, D., Lohman, D.J., Sodhi, N.S., Ng, P.K.L., Meier, R., Winker, K., Ingram, K.K., Das, I., 2007. Cryptic species as a window on diversity and conservation. Trends. Ecol. Evol. 22, 148-155. Bouckaert, R., Heled, J., Kuhnert, D., Vaughan, T., Wu, C.H., Xie, D., Suchard, M.A., Rambaut, A., Drummond, A.J., 2014. BEAST 2: A software platform for Bayesian evolutionary analysis. Plos Comput. Biol. 10. Cheviron, Z.A., Brumfield, R.T., 2009. Migration-selection balance and local adaptation of mitochondrial haplotypes in Rufous-Collared Sparrows (Zonotrichia Capensis) along an elevational gradient. Evolution 63, 1593-1605. Chiang, S.C., Du, N., 1979. Fauna Sinica (China), Crustacea: freshwater Cladocera. Academia Sinica, Science Press, Peking. (In Chinese). Colbourne, J.K., Crease, T.J., Weider, L.J., Hebert, P.D.N., Dufresne, F., Hobaek, A., 1998. Phylogenetics and evolution of a circumarctic species complex (Cladocera : Daphnia pulex). Biol. J. Linn. Soc. 65, 347-365. Cornils, A., Wend-Heckmann, B., Held, C., 2017. Global phylogeography of Oithona similis s.l. (Crustacea, Copepoda, Oithonidae) - A cosmopolitan plankton species or a complex of cryptic lineages? Mol. Phylogenet. Evol. 107, 473-485. Costa, F., deWaard, J.R., Boutillier, J., Ratnasingham, S., Dooh, R., Hajibabaei, M., Hebert, P.D.N., 1997. Biological identifications through DNA barcodes: the case of the Crustacea. Can. J. Fish Aquat. Sci. 64, 272-295. Dabramo, L.R., 1980. Ingestion rate decrease as the stimulus for sexuality in populations of Moina macrocopa. Limnol. Oceanogr. 25, 422-429. Darling, K.F., Kucera, M., Wade, C.M., 2007. Global molecular phylogeography reveals persistent Arctic circumpolar isolation in a marine planktonic protist. P. Natl. Acad. Sci. U.S.A. 104, 5002-5007. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772-772. deWaard, J.R., Sacherova, V., Cristescu, M.E., Remigio, E.A., Crease, T., Hebert, P.D.N., 2006. Probing the relationships of the branchiopod crustaceans. Mol. Phylogenet. Evol. 39, 491-502. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797. Elias-Gutierrez, M., Martinez-Jeronimo, F., Ivanova-Moreno, M., Hebert, P.D.N., 2008. DNA barcodes for Cladocera and Copepoda from Mexico and Guatemala, highlights and new discoveries. Zootaxa 1839, 1-42. Ezard, T., Fujisawa, T., Barraclough, T., 2009. Splits: Species’ Limits by Threshold Statistics, R package. Feulner, P.G.D., Kirschbaum, F., Schugardt, C., Ketmaier, V., Tiedemann, R., 2006. Electrophysiological and molecular genetic evidence for sympatrically occuring cryptic species in African weakly electric fishes (Teleostei : Mormyridae : Campylomormyrus). Mol. Phylogenet. Evol. 39, 198-208. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294-299. Forro, L., Korovchinsky, N.M., Kotov, A.A., Petrusek, A., 2008. Global diversity of cladocerans (Cladocera; Crustacea) in freshwater. Hydrobiologia 595, 177-184. Franco, F.F., Lavagnini, T.C., Sene, F.M., Manfrin, M.H., 2015. Mito-nuclear discordance with evidence of shared ancestral polymorphism and selection in cactophilic species of Drosophila. Biol. J. Linn. Soc. 116, 197-210. Funk, D.J., Omland, K.E., 2003. Species-level paraphyly and polyphyly: Frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Evol. S. 34, 397-423. Gompert, Z., Forister, M.L., Fordyce, J.A., Nice, C.C., 2008. Widespread mito-nuclear discordance with evidence for introgressive hybridization and selective sweeps in Lycaeides. Mol. Ecol. 17, 5231-5244. Goulden, C., 1968. The systematics and evolution of the Moinidae. Trans. Am. Philos. Soc. 58, 1-101. 20

Grosvenor, G., Smith, G., 1913. The life cycle of Moina rectirostris. Q. J. Microsc. Sci. 58, 511-522. Havel, J.E., Shurin, J.B., 2004. Mechanisms, effects, and scales of dispersal in freshwater zooplankton. Limnol. Oceanogr. 49, 1229-1238. Hebert, P.D.N., 1985. Interspecific hybridization between cyclic parthenogens. Evolution 39, 216-220. Hebert, P.D.N., Beaton, M.J., Schwartz, S.S., Stanton, D.J., 1989. Polyphyletic origins of asexuality in Daphnia pulex. I. Breeding-system variation and levels of clonal diversity. Evolution 43, 1004-1015. Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. P. Natl. Acad. Sci. U.S.A. 101, 14812-14817. Hudec, I., 1990. Moina-Weismanni Ishikawa, 1896 (Cladocera, Moinidae) in Central Europe. Hydrobiologia 190, 33-42. Jeffery, N.W., Elias-Gutierrez, M., Adamowicz, S.J., 2011. Species diversity and phylogeographical affinities of the Branchiopoda (Crustacea) of Churchill, Manitoba, Canada. PLOS One 6. Jensen, J.L., Bohonak, A.J., Kelley, S.T., 2005. Isolation by distance, web service. BMC Genet. 6. Jia, J.Y., Liu, X.J., Li, L., Lei, C.Q., Dong, Y., Wu, G.Q., Hu, G.F., 2018. Transcriptional and translational relationship in environmental stress: RNAseq and ITRAQ proteomic analysis between sexually reproducing and parthenogenetic females in Moina micrura. Front. Physiol. 9. Keller, B., Wolinska, J., Manca, M., Spaak, P., 2008. Spatial, environmental and anthropogenic effects on the taxon composition of hybridizing Daphnia. Philos. T. R. Soc. B. 363, 2943-2952. Kotov, A.A., Elias-Gutierrez, M., Granados-Ramirez, J.G., 2005. Moina dumonti sp nov (Cladocera, Anomopoda, Moinidae) from southern Mexico and Cuba, with comments on moinid limbs. Crustaceana 78, 41-57. Kotov, A.A., Forro, L., Korovchinsky, N.M., Petrusek, A., 2013. World checklist of freshwater Cladocera species. http://fada.biodiversity.be/group/show/17. Kotov, A.A., Taylor, D.J., 2011. Mesozoic fossils (> 145 Mya) suggest the antiquity of the subgenera of Daphnia and their coevolution with chaoborid predators. BMC Evol. Biol. 11. Kwak, K., Ji, K., Kho, Y., Kim, P., Lee, J., Ryu, J., Choi, K., 2018. Chronic toxicity and endocrine disruption of naproxen in freshwater waterfleas and fish, and steroidogenic alteration using H295R cell assay. Chemosphere 204, 156-162. Lampert, W., Sommer, U., 1999. Limnoökologie. Georg Thieme Verlag, Suttgart, New York. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451-1452. Linnen, C.R., Farrell, B.D., 2007. Mitonuclear discordance is caused by rampant mitochondrial introgression in Neodiprion (hymenoptera : diprionidae) sawflies. Evolution 61, 1417-1438. Liu, P., Xu, L., Xu, S.L., Martinez, A., Chen, H., Cheng, D., Dumont, H.J., Han, B.P., Fontaneto, D., 2018. Species and hybrids in the genus Diaphanosoma Fischer, 1850 (Crustacea: Branchiopoda: Cladocera). Mol. Phylogenet. Evol. 118, 369-378. Ma, X., Hu, W., Smilauer, P., Yin, M., Wolinska, J., In press. Daphnia galeata and D. dentifera are geographically and ecologically separated whereas their hybrids occur in intermediate habitats: survey of 44 Chinese lakes. Mol. Ecol. Doi: org/10.1111/mec.14991. Mangas-Ramirez, E., Sarma, S.S.S., Nandini, S., 2002. Combined effects of algal (Chlorella vulgaris) density and ammonia concentration on the population dynamics of Ceriodaphnia dubia and Moina macrocopa (Cladocera). Ecotox. Environ. Safe 51, 216-222. Marrone, F., Lo Brutto, S., Hundsdoerfer, A.K., Arculeo, M., 2013. Overlooked cryptic endemism in copepods: Systematics and natural history of the calanoid subgenus Occidodiaptomus Borutzky 1991 (Copepoda, Calanoida, Diaptomidae). Mol. Phylogenet. Evol. 66, 190-202. Mckay, B.D., Zink, R.M., 2010. The causes of mitochondrial DNA gene tree paraphyly in birds. Mol. Phylogenet. Evol. 54, 647-650. Mirabdullaev, I.M., 1993. Moina Gouldeni n. sp. (Cladocera, Moinidae) from Central-Asia. Crustaceana 64, 192-196.

21

Mirabdullayev, I.M., 1998. Moina mukhamedievi n. sp. (Crustacea, Cladocera) from ricefields of Uzbekistan (central Asia). Hydrobiologia 385, 11-16. Nedli, J., De Meester, L., Major, A., Schwenk, K., Szivak, I., Forro, L., 2014. Salinity and depth as structuring factors of cryptic divergence in Moina brachiata (Crustacea: Cladocera). Fund. Appl. Limnol. 184, 69-85. Padhye, S.M., Dumont, H.J., 2014. Moina hemanti sp nov., a new species of the genus Moina s.l. (Branchiopoda: Anomopoda) from Pune, India. Zootaxa 3860, 561-570. Pavlova, A., Amos, J.N., Joseph, L., Loynes, K., Austin, J.J., Keogh, J.S., Stone, G.N., Nicholls, J.A., Sunnucks, P., 2013. Perched at the mito-nuclear crossroads: divergent mitochondrial lineages correlate with environment in the face of ongoing nuclear gene flow in an Australian bird. Evolution 67, 3412-3428. Penton, E.H., Hebert, P.D.N., Crease, T.J., 2004. Mitochondrial DNA variation in North American populations of Daphnia obtusa: continentalism or cryptic endemism? Mol. Ecol. 13, 97-107. Petrusek, A., Cerny, M., Audenaert, E., 2004. Large intercontinental differentiation of Moina micrura (Crustacea : Anomopoda): one less cosmopolitan cladoceran? Hydrobiologia 526, 73-81. Petrusek, A., Thielsch, A., Schwenk, K., 2012. Mitochondrial sequence variation suggests extensive cryptic diversity within the Western Palearctic Daphnia longispina complex. Limnol. Oceanogr. 57, 1838-1845. Pfenninger, M., Schwenk, K., 2007. Cryptic animal species are homogeneously distributed among taxa and biogeographical regions. BMC Evol. Biol. 7. Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S., Kamoun, S., Sumlin, W.D., Vogler, A.P., 2006. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst. Biol. 55, 595-609. Prosser, S., Martinez-Arce, A., Elias-Gutierrez, M., 2013. A new set of primers for COI amplification from freshwater microcrustaceans. Mol. Ecol. Resour. 13, 1151-1155. R Development Core Team, 2009. R: a language and environment for statistical computing. R foundation for statistical computing, Vienna, Austria. Rambaut, A., Drummond, A., Xie, D., Baele, G., Suchard, M., 2018. Tracer v1.7, Available from http://beast.community/tracer. Rellstab, C., Keller, B., Girardclos, S., Anselmetti, F.S., Spaak, P., 2011. Anthropogenic eutrophication shapes the past and present taxonomic composition of hybridizing Daphnia in unproductive lakes. Limnol. Oceanogr. 56, 292-302. Salzburger, W., Ewing, G.B., von Haeseler, A., 2011. The performance of phylogenetic algorithms in estimating haplotype genealogies with migration. Mol. Ecol. 20, 1952-1963. Schwenk, K., Spaak, P., 1995. Evolutionary and ecological consequences of interspecific hybridization in cladocerans. Experientia 51, 465-481. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. Taylor, D.J., Sprenger, H.L., Ishida, S., 2005. Geographic and phylogenetic evidence for dispersed nuclear introgression in a daphniid with sexual propagules. Mol. Ecol. 14, 525-537. Thielsch, A., Knell, A., Mohammadyari, A., Petrusek, A., Schwenk, K., 2017. Divergent clades or cryptic species? Mito-nuclear discordance in a Daphnia species complex. BMC Evol. Biol. 17. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. Toews, D.P.L., Brelsford, A., 2012. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21, 3907-3930. Xia, X.H., 2013. DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution. Mol. Biol. Evol. 30, 1720-1728. Xia, X.H., Xie, Z., Salemi, M., Chen, L., Wang, Y., 2003. An index of substitution saturation and its application. Mol. Phylogenet. Evol. 26, 1-7.

22

Xu, S., Innes, D.J., Lynch, M., Cristescu, M.E., 2013. The role of hybridization in the origin and spread of asexuality in Daphnia. Mol. Ecol. 22, 4549-4561. Yin, M., Giessler, S., Griebel, J., Wolinska, J., 2014. Hybridizing Daphnia communities from ten neighbouring lakes: spatio-temporal dynamics, local processes, gene flow and invasiveness. BMC Evol. Biol. 14:80. Yin, M., Wang, X., Ma, X., Giessler, S., Petrusek, A., Griebel, J., Hu, W., Wolinska, J., 2018. Cytonuclear diversity and shared mitochondrial haplotypes among Daphnia galeata populations separated by seven thousand kilometres. BMC Evol. Biol. 18:130. Yin, M., Wolinska, J., Giessler, S., 2010. Clonal diversity, clonal persistence and rapid taxon replacement in natural populations of species and hybrids of the Daphnia longispina complex. Mol. Ecol. 19, 41684178. Zhang, J.J., Kapli, P., Pavlidis, P., Stamatakis, A., 2013. A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29, 2869-2876.

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Table legends: Table 1. List of localities inhabited by Moina (name, abbreviation and geographical position) and genetic characterization of sequenced individuals. The capital letters in the COI clade column indicate lineages, which were reported in previous studies, and the lowercase Roman numerals indicate new lineages from China.

Table 2. The list of all ITS-1 alleles. Bold type indicates the mismatch assignment by COI and ITS-1. Abbreviations of populations are provided in Table 1.

Table S1. List of reference COI sequences of Moina species from the Central Siberian Plateau, the East European Plain, East Siberia and the Western Siberian Plain of Russia, Hungary, Kazakhstan, Mongolia, Ukraine and Tibet of China. All these reference sequences were from Bekker et al., (2016).

Table S2. List of reference COI sequences of Moina species from Hungary, China, Russia, South Korea, Mexico and Canada. References: (a) Nedli et al., direct submission, (b) Chen et al., direct submission, (c) Frolova et al., direct submission, (d) Prosser et al., (2013), (e) Elías-Gutiérrez et al., (2008), (f) deWaard et al., (2006), (g) Costa et al., (2007) and (h) Jeffery et al., (2011).

24

Figure legends: Figure 1. Distribution of lineages of Moina in Eurasia based on COI. Solid black circles indicate habitats with Moina in China, empty black circles indicate the habitats with Moina from Kotov (2010), and solid grey circles indicate localities at which Moina was not detected. Country names are abbreviated as: CN: China, HU: Hungary, KZ: Kazakhstan, MN: Mongolia, RU: Russia and UA: Ukraine. Lineage IDs are in open circles and listed in Table 1 and S1.

Figure 2. Bayesian phylogenetic tree and lineage delimitation results for Moina, based on the mitochondrial COI gene (478 bp). A single representative of each haplotype (including all haplotypes represented in GenBank) was included in the tree. Codes of Moina individuals from China are provided in Table 1; for origin of reference sequences’ IDs see Tables S1 and S2. Only posterior probabilities > 0.70 are shown. The lineage IDs are shown in circles, and newly reported ones indicated in Roman numerals. Circles with a pink background indicate the 11 lineages detected in China. Lineage delimitation according to the GMYC and bPTP methods are indicated. For the bPTP method, the statistical support (PP) for clade membership is also shown. Abbreviations of country names in which each haplotype was detected are, CAN: Canada, CN: China, HU: Hungary, KZ: Kazakhstan, KOR: South Korean, MEX: Mexico, MN: Mongolia, RU: Russia and UA: Ukraine. Taxon names follow Bekker et al., (2016). Ceriodaphnia was used as an outgroup.

Figure 3. Relative timing of the diversification of Moina based on COI sequences. Only posterior probabilities > 0.70 are shown. Blue horizontal bars at nodes indicate the 95% HPD intervals of

25

clade ages. The lineage IDs are shown in circles, and newly reported ones indicated in Roman numerals. Circles with a pink background colour indicate the 11 lineages detected in China.

Figure 4. Haplotype network of Moina, based on the mitochondrial COI gene (478 bp). Each circle represents a unique haplotype and its size reflects the number of individuals carrying that haplotype. Colour codes allow easy discrimination of regions in the network. Segment sizes within circles indicate the distribution of haplotypes among different regions. The lineage IDs are shown in open circles, and newly reported ones indicated in Roman numerals. Haplotypes of the same species (based on the phylogeny) are framed with red lines, and the species names given. The number of marks on connecting lines indicates the number of mutations separating haplotypes.

Figure 5. Scatterplot of pairwise geographical distance (kilometres) versus genetic distance (FST based on COI) among (a) M. cf. brachiata, (b) M. cf. micrura and (c) M. cf. macrocopa populations.

Figure 6. The Bayesian phylogenetic tree of the ITS-1 gene (636 bp) of Moina lineages from China. The taxon names were given based on the COI phylogeny. Only posterior probabilities > 0.70 are shown. Lineage delimitation according to the GMYC and bPTP methods are indicated. For the bPTP method, the statistical support (PP) for clade membership is also shown. These lineage IDs are shown with Greek letters in circles. The list of ITS-1 alleles is provided in Table 2. The mismatch assignment by COI and ITS-1 are labelled with bold, see Table 2.

26

Figure S1. Morphology of adult female Moina species. (A) M. cf. brachiata; (B) M. cf. micrura; (C) M. cf. macrocopa and (D) M. cf. salina. Black arrows indicate the supraocular depression on the head and the elevation of dorsum of valves. Black crosses indicate the absence of the supraocular depression on the head or the elevation of dorsum of valves.

Figure S2. The body length across M. cf. macrocopa, M. cf. brachiata, M. cf. salina and M. cf. micrura.

Figure S3. Maximum likelihood tree representing the diversity among phylogroups of Moina, based on the mitochondrial COI gene (454 bp). Sequences of Moina individuals in this study were provided in Table 1 and the reference sequences (including all published sequences represented in GenBank) were provided in Table S1 and S2. Only posterior probabilities > 0.70 are shown.

Figure S4. Maximum likelihood tree representing the diversity among phylogroups of M. cf. brachiata, based on the mitochondrial COI gene (484 bp). Sequences of Moina cf. brachiata in this study were provided in Table 1 and all the reference sequences of Moina cf. brachiata were provided in Table S1 and S2. Only posterior probabilities > 0.70 are shown.

Figure S5. Maximum likelihood tree representing the diversity among phylogroups of M. cf. macrocopa, based on the mitochondrial COI gene (504 bp). Sequences of Moina cf. macrocopa in this study were provided in Table 1 and all the reference sequences of Moina cf. macrocopa were provided in Table S1 and S2. Only posterior probabilities > 0.70 are shown.

27

Table 1. List of localities inhabited by Moina (name, abbreviation and geographical position) and genetic characterization of sequenced individuals. The capital letters in the COI clade column indicate lineages, which were reported in previous studies, and the lowercase Roman numerals indicate new lineages from China.

Lake (abbreviation)

Latitude, longitud e

Mitochondrial gene Morpholog

mt DNA

y Taxon

Taxon

Nuclear gene

COI

ITS H

π

0.71

0.0816

1

4

0.82

0.0408

1

7

0.85

0.0593

ⅲ,

7

7

ⅳ

MSM2

0

0

1

MSM2

0

0

8

3

MSI2, GYH1, GYH2

0.46

0.0010

4

5

12

1

MSI4

0

0

N1

N2

Haplotype

10

4

MSI3, MSI4, MSI5, MSI6

8

4

MSI5, MSI6, MSI7, DON1

7

5

MSI2, MSI3, MSI6, MSI8, GEH1

11

1

11

COI

Clade

N3

N4

ⅳ, I

1

2

I

ⅳ, I

2

2

I

0

0

ⅵ

1

2

ⅵ

ⅵ

2

3

ⅵ

ⅲ

0

0

I

0

0

clade

Eastern Plain Baijia Lake

31.93N,

M. cf.

M. cf.

(BJH)

119.14E

micrura

micrura

Dong Lake

30.57N,

M. cf.

M. cf.

(DON)

114.38E

micrura

micrura

31.62N,

M. cf.

M. cf.

119.83E

micrura

micrura

Gejiarenzu Pond

31.57N,

M. cf.

M. cf.

(GJP)

119.38E

macrocopa

macrocopa

Gejianiutou Pond

31.58N,

M. cf.

M. cf.

(GNP)

119.39E

macrocopa

macrocopa

Gaoyou Lake

32.53N,

M. cf.

M. cf.

(GYH)

119.15E

micrura

micrura

Jinshui Lake

26.13N,

M. cf.

M. cf.

(JSL)

119.10E

micrura

micrura

Ge Lake (GEH)

Leqing Lake

28.14N,

M. cf.

M. cf.

(LQH)

120.94E

macrocopa

macrocopa

30.76N,

M. cf.

M. cf.

120.76E

micrura

micrura

M. cf.

M. cf.

micrura

micrura

Nan Lake (NAH)

Poyang Lake

29.16N,

(PYH)

116.27E

M. cf.

M. cf.

macrocopa

macrocopa

Qingshan Lake

30.24N,

M. cf.

M. cf.

(QSH)

119.77E

micrura

micrura

Qinting Lake

26.15N,

M. cf.

M. cf.

(QTH)

119.29E

micrura

micrura

Shijiu Lake

31.46N,

M. cf.

M. cf.

(SJH)

118.86E

micrura

micrura

30.25N,

M. cf.

M. cf.

120.16E

micrura

micrura

32.62N,

M. cf.

M. cf.

108.90E

micrura

micrura

Xi Lake (XIH) Ying Lake (YIH)

MSM2

0

0

ⅵ

1

1

ⅵ

MSI3, MSI4, MSI6, MSI8, NAH1, NAH2,

0.91

0.0568

ⅳ,

NAH3

7

9

ⅴ, I

1

1

ⅴ

0.75

0.0044

0

2

I

0

0

0

0

ⅵ

0

0

0.78

0.0620

ⅲ,

2

8

ⅳ

1

2

ⅲ

0

0

ⅳ

1

1

ⅳ

0.20

0.0004

0

2

ⅳ

0

0

0.73

0.0645

ⅲ,

3

7

ⅳ

0

0

MSI2

0

0

ⅲ

0

0

1

DAH1

n.s

n.s

B

0

0

11

2

MSI4, MSI7

0.18

0.0007

2

6

I

0

0

12

2

MSI1, MSI2

0.48

0.0010

5

1

ⅲ

0

0

11

1

9

7

9

4

MSI4, MSI5, PYH1, PYH2

1

1

MSM2

11

4

MSI2, MSI3, MSI6, MSI11

10

1

MSI8

10

2

MSI6, MSI8

10

3

MSI2, MSI6, XIH1

6

1

1

Inner Mongolia-Xinjiang Plateau 40.58N,

M. cf.

M. cf.

112.70E

brachiata

brachiata

40.61N,

M. cf.

M. cf.

110.97E

micrura

micrura

Amuta Pond

46.51N,

M. cf.

M. cf.

(AMT)

124.25E

micrura

micrura

Daihai (DAH) Hasuhai (HSH) Northeast Plain

Donghu

46.63N,

M. cf.

M. cf.

Reservoir (DOH)

125.61E

micrura

micrura

48.80N,

M. cf.

M. cf.

124.26E

brachiata

brachiata

Huoshaoli Pond

46.66N,

M. cf.

M. cf.

(HSL)

124.04E

micrura

micrura

M. cf.

M. cf.

45.87N,

brachiata

brachiata

Dumeng (DUM)

Kuli Pond (KLP)

124.82E

M. cf.

M. cf.

micrura

micrura

M. cf.

M. cf.

Lianhuan Lake

46.83N,

brachiata

brachiata

(LHH)

124.40E

M. cf.

M. cf.

micrura

micrura

Dalonghu Pond

46.81N,

M. cf.

M. cf.

(LHP)

124.32E

micrura

micrura

Liming Lake

46.59N,

M. cf.

M. cf.

(LIM)

125.12E

micrura

micrura

Nanyin

45.95N,

M. cf.

M. cf.

Reservoir (NYR)

124.58E

micrura

micrura

46.80N,

M. cf.

M. cf.

124.25E

micrura

micrura

Qingken Pond

46.39N,

M. cf.

M. cf.

(QKP)

125.45E

micrura

micrura

Renmin Pond

44.58N,

M. cf.

M. cf.

(RMP)

129.63E

micrura

micrura

Qijia Pond (QJP)

0.46

0.0009

7

8

MSB4, MSB5, MSB6, DUM1, DUM2,

0.83

0.0542

DUM3

3

8

0.50

0.0010

9

7

0.60

0.0012

0

6

0.86

0.0043

7

2

0.60

0.0012

0

6

0.40

0.0025

0

1

0.60

0.0439

0

0

0

0

0.73

0.0290

3

6

0.71

0.0696

2

4

0.75

0.0046

8

3

1.00

0.0020

0

9

10

2

MSI1, MSI2

9

6

11

2

MSI1, MSI2

5

2

MSB6, KLP1

6

4

MSI4, MSI5, MSI9, MSI10

5

2

MSB6, LHH1

5

2

MSI4, MSI5

11

4

MSI1, MSI2, MSI5, MSI10

10

1

MSI1

10

4

MSI1, MSI2, MSI4, NYR1

12

3

MSI1, MSI2, MSI5

12

4

MSI4, MSI5, MSI10, QKP1

2

2

MSI4, RMP1

ⅲ

0

0

B, G

3

2

ⅲ

0

0

G

1

2

I

0

0

G

2

2

I

0

0

ⅲ, I

0

0

ⅲ

0

0

ⅲ, I

0

0

ⅲ, I

0

0

I

0

0

I

0

0

B

G

G

Songhuajiang

45.70N,

M. cf.

M. cf.

Pond (SHJ)

124.78E

micrura

micrura

Talahong Pond

46.77N,

M. cf.

M. cf.

(TLH)

124.22E

micrura

micrura

M. cf.

M. cf.

brachiata

brachiata

Xidahai nearby

45.96N,

(XDF)

124.66E

Xidahai (XDP)

M. cf.

M. cf.

salina

salina

M. cf.

M. cf.

brachiata

brachiata

46.06N,

M. cf.

M. cf.

124.60E

micrura

micrura

M. cf.

M. cf.

salina

salina

M. cf.

M. cf.

Xinhua Lake

46.14N,

brachiata

brachiata

(XHP)

124.62E

M. cf.

M. cf.

micrura

micrura

0.71

0.0040

1

9

0.53

0.0011

0

1

0.85

0.0659

7

5

1.00

0.0034

0

9

0.63

0.0754

9

3

MSI5

0

1

XDP1

11

3

MSB6, XHP1, XHP2

1

1

MSI10

10

2

MSB1, MSB2

12

1

10

1

10

3

MSI4, MSI5, MSI9

I

2

2

12

2

MSI1, MSI2

7

4

MSB4, MSB5, MSB6, XDF3

4

4

XDF1, XDF2, XDF4, XDF5

9

3

MSB4, MSB6, XDP2

1

1

1

I

ⅲ

0

0

B, G

2

2

B, G

N

1

1

N

B, G

1

2

B

0

I

0

0

0

0

N

0

0

0.65

0.0016

5

0

G

3

3

0

0

I

0

0

0.46

0.0185

ⅰ,

7

5

ⅱ

3

3

ⅰ

MSB3

0

0

ⅱ

3

2

ⅱ

MSB3

0

0

ⅱ

3

2

ⅱ

G

Qinghai-Tibet Plateau Caicuo 1 Pond

28.79N,

M. cf.

M. cf.

(C1C)

90.47E

brachiata

brachiata

Dongla Pond

29.00N,

M. cf.

M. cf.

(D1L)

90.86E

brachiata

brachiata

Dongla 2 Pond

29.01N,

M. cf.

M. cf.

(D2L)

90.85E

brachiata

brachiata

Gongbuxue Lake

28.84N,

M. cf.

M. cf.

(GBX)

91.03E

macrocopa

macrocopa

M. cf.

M. cf.

Muchang 1 Pond

28.93N,

brachiata

brachiata

(M1C)

91.06E

M. cf.

M. cf.

macrocopa

macrocopa

Qinghai Lake

36.82N,

M. cf.

M. cf.

(QHH)

100.35E

salina

salina

Ruifeng 1 Pond

28.92N,

M. cf.

M. cf.

(R1F)

91.05E

macrocopa

macrocopa

Ruifeng 2 Pond

28.87N,

M. cf.

M. cf.

(R2F)

91.07E

macrocopa

macrocopa

M. cf.

M. cf.

Ruifeng 3 Pond

28.86N,

brachiata

brachiata

(R3F)

91.09E

M. cf.

M. cf.

macrocopa

macrocopa

M. cf.

M. cf.

Sangzhu Lake

28.77N,

brachiata

brachiata

(SZP)

90.65E

M. cf.

M. cf.

macrocopa

macrocopa

Tebula Lake

28.81N,

M. cf.

M. cf.

(TBL)

90.74E

brachiata

brachiata

Xincuo 3 Pond

29.99N,

M. cf.

M. cf.

(X3C)

94.19E

macrocopa

macrocopa

Yigong Pond

30.15N,

M. cf.

M. cf.

(YGC)

95.00E

macrocopa

macrocopa

0

0

ⅵ

2

1

ⅵ

1.00

0.0355

ⅰ,

0

6

ⅱ

1

2

ⅰ

0.50

0.0010

0

5

ⅵ

1

1

ⅵ

QHH1

0

0

O

2

3

O

1

MSM1

0

0

ⅵ

1

1

ⅵ

11

1

MSM1

0

0

ⅵ

0

0

5

1

MSB7

0

0

ⅱ

3

2

3

1

MSM1

0

0

ⅵ

0

0

11

2

MSB1, MSB2

0.32

0.0130

ⅰ,

7

1

ⅱ

1

1

1

1

MSM1

0

0

ⅵ

0

0

12

2

MSB1, TBL1

0.16

0.0003

7

5

ⅰ

2

2

10

1

MSM1

0

0

ⅵ

0

0

12

2

MSM1, YGC1

0.16

0.0027

7

9

ⅵ

0

0

12

1

MSM1

2

2

MSB1, MSB7

9

2

MSM1, M1C1

11

1

10

ⅱ

ⅱ

ⅰ

Yanghu 1 Pond

29.16N,

M. cf.

M. cf.

(Y1P)

90.77E

brachiata

brachiata

Yanghu nearby

29.12N,

M. cf.

M. cf.

(YHB)

91.75E

brachiata

brachiata

24.30N,

M. cf.

M. cf.

102.42E

micrura

micrura

12

1

MSB8

0

0

ⅱ

0

0

12

1

MSB8

0

0

ⅱ

3

2

11

2

MSI4, MSI5

0.32

0.0020

7

5

I

0

0

ⅱ

Yunnan-Guizhou Plateau Cui Lake (CUH)

N1 is the number of individuals used for COI sequencing, N2 is the number of haplotypes, H is haplotype diversity, π is nucleotide diversity, N3 is the number of individuals for ITS-1 sequencing, and N4 is the number of sequences.

Table 2. The list of all ITS-1 alleles. Bold type indicates the mismatch assignment by COI and ITS-1. Abbreviations of populations are provided in Table 1. ITS-1 Allele ID

mtDNA lineage ID

M. cf. brachiata I-1

M. cf. brachiata lineage B

XDP2-b

M. cf. brachiata lineage B

DUM1/DUM2/DUM4-a/XDF2/XDP2-a

M. cf. brachiata lineage G

XHP3

M. cf. brachiata lineage B

DUM4-b

M. cf. brachiata lineage ⅰ

C1C1-a/M1C3-b/TBL1-a

M. cf. brachiata lineage ⅱ

D1L1-a/D1L2-a/D1L3-a/D2L1-a/D2L2-a/D2L3-a/R3F1-b/R3F12-a/SZP4/YHB1-b/YHB2/YHB3-b

M. cf. micrura lineage ⅲ

QSH12-a

M. cf. brachiata lineage ⅰ

C1C1-b/C1C5/TBL1-b/TBL2

M. cf. brachiata lineage ⅱ

D1L1-b/D1L2-b/D1L3-b/D2L1-b/D2L2-b/D2L3-b

M. cf. micrura lineage ⅲ

QSH12-b

M. cf. brachiata lineage ⅰ

C1C8/M1C3-a

M. cf. brachiata lineage ⅱ

R3F1-a/R3F9/R3F12-b/YHB1-a/YHB3-a

M. cf. brachiata I-7

M. cf. brachiata lineage G

KLP3-a/LHH1-a/LHH2-a/XDF4/XHP2-a/XHP4-a

M. cf. brachiata I-8

M. cf. brachiata lineage G

KLP3-b/LHH1-b/LHH2-b/XHP2-b/XHP4-b

M. cf. micrura I-1

M. cf. micrura lineage I

BJH6-a

M. cf. micrura I-2

M. cf. micrura lineage I

DON1

M. cf. micrura I-3

M. cf. micrura lineage I

DON4

M. cf. micrura I-4

M. cf. micrura lineage I

SHJ5

M. cf. micrura I-5

M. cf. micrura lineage I

BJH6-b

M. cf. micrura I-6

M. cf. micrura lineage I

SHJ4

M. cf. brachiata I-2 M. cf. brachiata I-3 M. cf. brachiata I-4

M. cf. brachiata I-5

M. cf. brachiata I-6

ITS-1 sequence IDs

M. cf. micrura I-7

M. cf. micrura lineage ⅳ

QTH1

M. cf. micrura I-8

M. cf. micrura lineage ⅴ

NAH8

M. cf. macrocopa I-1

M. cf. macrocopa lineage ⅵ

GNP1-b/M1C5

M. cf. macrocopa I-2

M. cf. macrocopa lineage ⅵ

GJP17-b/GNP3-b

M. cf. macrocopa I-3

M. cf. macrocopa lineage ⅵ

GBX2/GBX9/GJP17-a/GNP1-a/GNP3-a/LQH1/ R1F2

M. cf. salina I-1

M. cf. salina lineage N

XDF11

M. cf. salina I-2

M. cf. salina lineage O

QHH1-b

M. cf. salina I-3

M. cf. salina lineage O

QHH1-a/QHH2-a

M. cf. salina I-4

M. cf. salina lineage O

QHH2-b

• • • •

We explored lineage diversity and regional distribution of Moina in China Four Moina species complexes with eleven mtDNA lineages occurred across China Discordance between mtDNA and nuclear ITS-1 phylogenies of Moina was detected An mtDNA phylogeny showed apparent paraphyly in two Moina taxa

28