Hydrobiologia (2020) 847:1651–1661 https://doi.org/10.1007/s10750-019-04110-2 (0123456789().,-volV)( 0123456789().,-volV) TRENDS IN AQUATICECOLOGYIII Are Cardinium infections causing asexuality in non-marine ostracods? Isa Scho¨n . Koen Martens Received: 28 June 2019 / Revised: 6 October 2019 / Accepted: 17 October 2019 / Published online: 14 November 2019 Ó Springer Nature Switzerland AG 2019 Abstract Endosymbiotic bacteria manipulating host is a significant positive correlation between latitude biology and reproduction, and sometimes also causing and Cardinium infection, which might be linked to parthenogenesis, are known from many metazoan geographic parthenogenesis, a common phenomenon taxa. Three recent studies have reported Cardinium in non-marine ostracods with mixed reproduction. We endosymbionts in non-marine ostracods with different suggest that the observed patterns best fit a polymor- reproductive modes. Here, we test with all available phic equilibrium between endosymbionts and their data which (a)biotic factors could possibly shape hosts. Ostracods with mixed reproduction often pro- infection patterns in these crustaceans. The presence duce young asexual lineages, implying that Cardinium of Cardinium in non-marine ostracods differs signif- infections might have occurred more recently, and are icantly between genders and between species with widespread. In contrast, putative ancient asexual different reproductive modes. We observed more darwinulid ostracod shows less frequent occurrence infections in females and found Cardinium only in of Cardinium. Loss of endosymbionts in these asexual ostracods with mixed and asexual reproduction. There ostracods during their long evolutionary histories of millions of years seems a more likely explanation. Which factors influence Cardinium prevalence in non- Guest editors: Koen Martens, Sidinei M. Thomaz, marine ostracods needs to be further tested in life Diego Fontaneto & Luigi Naselli-Flores / Emerging history experiments. Trends in Aquatic Ecology III. Electronic supplementary material The online version of Keywords Endosymbiont Á Parthenogenesis Á Mixed this article (https://doi.org/10.1007/s10750-019-04110-2) con- reproduction Á 16S tains supplementary material, which is available to authorized users. I. Scho¨n(&) Á K. Martens Introduction Royal Belgian Institute of Natural Sciences, OD Nature, Freshwater Biology, Brussels, Belgium The paradox of sex remains one of the last open e-mail: [email protected] evolutionary questions. All present-day asexual ani- I. Scho¨n mal populations or species have originated from Research Group Zoology, University of Hasselt, sexual ancestors at one point in their history (Scho¨n Diepenbeek, Belgium et al., 2009a). How asexuality originates and is K. Martens maintained is central for our understanding of genetic Dept Biology, University of Ghent, Ghent, Belgium diversity and adaptability in asexuals, which in turn 123 1652 Hydrobiologia (2020) 847:1651–1661 determines the evolutionary fate of asexuals in the Mixed reproduction is particularly common in long and short terms. Infections with endosymbionts another group of non-marine ostracods, the family are one of the major drivers for the transition from sex Cyprididae (Martens, 1998), which comprises half of to asexuality in invertebrates (Hilgenboecker et al., the more than 2300 known extant non-marine ostracod 2008; Engelsta¨dter & Hurst, 2009; Zug & Hammer- morphospecies (Meisch et al., 2019). Mixed repro- stein, 2012; Ma & Schwander, 2017) and can induce duction in ostracods is very different from cyclic or parthenogenesis or cause female-biased sex ratios facultative parthenogenesis as known from cladocer- through feminization and male killing. Likewise, ans (Decaestecker et al., 2009), rotifers (Serra & Snell, endosymbionts have been shown to maintain the co- 2009) or aphids (Simon et al., 2002) because mixed existence of sexual (non-infected) and asexual (in- reproduction in ostracods usually involves three fected) host strains, thus causing reproductive poly- ‘‘genders’’: males, sexual females and asexual females morphisms or mixed reproduction (Ma & Schwander, (Martens, 1998). In contrast to cyclic parthenogenesis, 2017). Endosymbiotic infections can have profound asexuality in non-marine ostracods is irreversible effects on the microevolution and reproductive ecol- (Martens, 1998). Asexual and sexual females, which ogy of their hosts (Engelsta¨dter & Hurst, 2009). Some are morphologically indistinguishable, can occur of the best studied endosymbionts are bacteria of the sympatrically (Adolfsson et al., 2010; Bode et al., genus Wolbachia, which are known to be particularly 2010; Schmit et al., 2012). In Europe, mixed repro- widespread in terrestrial arthropods and can manipu- duction is frequently combined with geographic late the host’s reproductive system (Werren et al., parthenogenesis, where asexual ostracod populations 2008). A review (Ma & Schwander, 2017) revealed have a wide distribution, while sexuals are restricted to that 124 host species from seven different arthropod areas around the Mediterranean (Horne & Martens, taxa showed evidence for endosymbiont-induced 1999; Adolfsson et al., 2010; Schmit et al., 2012). parthenogenesis; Wolbachia was with 65–75% occur- Scho¨n et al. (2018) were the first to report rence the most frequent endosymbiont. Rickettsia, Cardinium in non-marine ostracods, investigating Arsenophonus, Cardinium and Spiroplasma can also more than 300 specimens from 22 morphospecies, affect reproductive modes and sex ratios in various followed by Mioduchowska et al. (2018) who found arthropods (Engelsta¨dter & Hurst, 2009; Van- Cardinium in the ostracod Heterocypris incongruens thournout et al., 2011; Ma & Schwander, 2017) but and C¸ elen et al. (2019) who screened 64 non-marine the exact mechanisms are less well understood for ostracod populations from Turkey representing 24 those endosymbionts. The currently available data on morphospecies. In light of these observations, we the presence of parthenogenesis-inducing endosym- further investigate here to what extent these endosym- bionts in invertebrates are still biased towards studies biotic infections could be the cause of parthenogenesis on terrestrial insects, especially Hymenoptera, and in non-marine ostracods. For this aim, we statistically probably underestimates the real prevalence of these reanalyse all existing data of Mioduchowska et al. bacteria in other (arthropod) groups (Ma & Schwan- (2018), Scho¨n et al. (2018) and C¸ elen et al. (2019)to der, 2017). test specifically for (1) differences in infection preva- Non-marine ostracods show a high incidence of lence between female and male non-marine ostracods; obligate parthenogenesis and multiple origins of (2) differences in the frequency of Cardinium infec- asexuality in different lineages (Chaplin et al., 1994; tions according to reproductive modes and type of Martens, 1998; Butlin et al., 1998). Although ancient habitat; (3) correlations between Cardinium infections asexuality of the non-marine Darwinulidae is still and latitude as a proxy for temperature effects. debated (Smith et al., 2006; Martens & Scho¨n, 2008; Scho¨n et al., 2009b), these ostracods currently remain one of the few examples of putative ancient asexual Materials and methods scandals (Heethoff et al., 2009; Mark Welch & Ricci, 2009; Scho¨n et al., 2009a, b; Schwander et al., 2011; Sampling and screening for Cardinium was done Schwander, 2016), having possibly reproduced exclu- differently in the three published reports on endosym- sively asexually for up to 200 myr (Martens et al., bionts of non-marine ostracods. Two studies (Scho¨n 2003). et al., 2018;C¸ elen et al., 2019) focused on non-marine 123 Hydrobiologia (2020) 847:1651–1661 1653 ostracods only, whereas Mioduchowska et al. (2018) pooled females as being infected, we counted this investigated a wider range of aquatic crustaceans for sample as a single data point for infected females. endosymbionts from temporary ponds and only one Secondly, we defined the reproductive mode at the ostracod species, Heterocypris incongruens. Scho¨n morphospecies level relying on the information in et al. (2018) screened non-marine ostracods from all Wouters (1988), Rossetti & Martens (1998), Meisch over the world, while C¸ elen et al. (2019) limited their (2000), and Martens (2001). We distinguished study to Turkey; both studies investigated non-marine between species with mixed reproduction and fully ostracods from four different types of habitats: sexual and asexual species. Species were regarded as springs, permanent water bodies (lakes, ponds, creeks fully sexual when males were present in all known and leaf litter), streams and temporary aquatic habitats populations. Similarly, species without any male (pools, wetlands, ditches or troughs). Scho¨n et al. records were considered as fully asexual. The presence (2018) also compared results of classic PCRs and of males in a population was regarded as evidence for Sanger sequencing with data of high-throughput sexual reproduction of that population (Martens, sequencing, while neither of the other two studies 1998). Species with male records from a single used such an approach. Furthermore, Scho¨n et al. geographic location only or species with geographic (2018) and Mioduchowska et al. (2018) screened parthenogenesis (where both sexual and asexual individual ostracods by classic PCR for the presence populations are known) were regarded as having of Cardinium but C¸ elen