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Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake “endemic” sponge lineages
Journal of Great Lakes Research xxx (xxxx) xxx
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Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages Dirk Erpenbeck a,b,⇑, Adrian Galitz a, Gert Wörheide a,b,c, Christian Albrecht d, Roberto Pronzato f, Renata Manconi e,f,⇑ a
Department of Earth & Environmental Sciences, Palaeontology and Geobiology, Ludwig-Maximilians-Universität München, Munich, Germany GeoBio-CenterLMU, Ludwig-Maximilians-Universität München, Munich, Germany SNSB – Bayerische Staatssammlung für Paläontologie und Geologie, Munich, Germany d Department of Animal Ecology and Systematics, Systematics and Biodiversity Group, Justus Liebig University Giessen, Giessen, Germany e ` di Genova, Genova, Italy Dipartimento di Scienze della Terra, dell’Ambiente e della Vita, Universita f Dipartimento Medicina Veterinaria, Università di Sassari, Sassari, Italy b c
a r t i c l e
i n f o
Article history: Received 3 May 2019 Accepted 16 September 2019 Available online xxxx Communicated by: Bjorn Stelbrink
Keywords: Freshwater sponges Spongillida Ancient lakes Ochridaspongia Cortispongilla Balliviaspongia
a b s t r a c t Spongillida (freshwater sponges) constitute an integral part of the world’s lacustrine ecosystems. Their radiation and worldwide distribution into nearly every freshwater habitat is predominantly facilitated by the formation of so-called gemmules, i.e., globular endurance and propagation stages. Widespread species produce gemmules in a wide array of morphs, while in (ancient) lake endemic lineages gemmules are absent. Contrary to current classification, earlier molecular studies on non-type material indicated that gemmule-lacking lineages are unrelated, and endemic taxa were derived from a cosmopolitan (and paraphyletic) founder lineage. In this study we investigate this hypothesis with type material, particularly focussing on endemic taxa potentially derived from common ancestor shared with the widespread Ephydatia fluviatilis. We demonstrate that taxa regarded as endemic from Lake Ohrid (Ochridaspongia), Lake Kinneret/Tiberias (Cortispongilla) and Lake Titicaca (Balliviaspongia) are derived from the founder lineage, but unrelated to other, particularly Afrotropical, ancient lake sponges, and discuss implications for understanding freshwater sponge phylogeny and evolution. Ó 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Introduction Freshwater sponges (Porifera: Demospongiae: Spongillida) are pivotal organisms in freshwater systems on all continents except Antarctica (Manconi and Pronzato, 2008). They have colonized almost every limnic habitat from lentic mountain lakes to brackish lotic estuaries (Manconi and Pronzato, 2007), from abyssal depths of ancient lakes (Crane et al., 1991; Itskovich et al., 2015) to surface regions, and above as ‘‘arboreal sponges” (see Manconi and Pronzato, 2016 for overview). Present-day freshwater sponges (order Spongillida Manconi & Pronzato) constitute a monophyletic group (e.g., Itskovich et al., 1999), implying that successful transition from the marine to brackish and freshwater habitats occurred
⇑ Corresponding authors at: Department of Earth & Environmental Sciences, Palaeontology and Geobiology, Ludwig-Maximilians-Universität München, Munich, Germany (D. Erpenbeck). Dipartimento Medicina Veterinaria, Università di Sassari, Sassari, Italy (R. Manconi). E-mail addresses: [email protected] (D. Erpenbeck), [email protected] (R. Manconi).
only once. Transition was followed by extensive dispersal and radiation to presently ~240 species (51 genera, Manconi and Pronzato, 2015; Pronzato et al., 2017), which exceeds numbers of other sessile freshwater filter feeders like Cnidaria and Bryozoa (Manconi and Pronzato, 2007). Key adaptive traits facilitating spongillid dispersal and radiation are high plasticity of body plan, physiology, life cycle and reproductive strategies, but particularly cryptobiosis by gemmules (Manconi and Pronzato, 2007). Gemmules constitute globular dormant resting bodies that may endure unfavourable abiotic conditions, including drought and freezing, which affect freshwater systems more frequently than marine (Frost et al., 1982; Pronzato and Manconi, 1995, 1994) Gemmules also facilitate easy dispersal as they can possess ‘‘pneumatic layers” for floating or potentially wind-driven dispersal, and/or possess a spiny outer theca to hook onto carriers such as birds (see overview in Manconi and Pronzato, 2007). Consequently, presence or absence of gemmules and differences in gemmular morphology is believed to be a key for dispersal success of spongillid lineages, facilitating nearly global distribution of the genera Spongilla Lamarck or (to a lesser extent) Ephydatia Lamouroux and Eunapius Gray (all family
https://doi.org/10.1016/j.jglr.2019.09.028 0380-1330/Ó 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Spongillidae Gray). Several other genera, however, apparently lack gemmule formation and remain strictly endemic to small spatial ranges (e.g., Cortispongilla Annandale to Lake Kinneret/Tiberias and River Jordan) (Manconi and Pronzato, 2002). Ancient lakes (here defined as lakes older than 130,000 years, i.e., existing for at least a glacial cycle, Hampton et al., 2018) are renownded for their invertebrate radiations (e.g., Genner et al., 2007) and harbour a wide range of freshwater sponge lineages. These include gemmule-lacking endemic taxa like Lubomirskiidae Weltner (Lake Baikal, four genera), and Metschnikowiidae Czerniavsky (Caspian Sea, monogeneric), but also Ochridaspongia Arndt (Lake Ohrid), Pachydictyum Weltner (Lake Poso), Spinospongilla Brien (Lake Tanganyika) and Malawispongia Brien (Lake Malawi) (Manconi and Pronzato, 2002). The latter genera are combined with Cortispongilla into the family Malawispongiidae Manconi and Pronzato, which was firstly erected as Globulospongillinae by Brien (1973) and Globulospongillidae by Racek (1974) to accommodate gemmule-lacking ancient lake taxa and now comprises five endemic, predominantly monotypic genera with a notably disjunct distribution (Manconi and Pronzato, 2002). While molecular data support the monophyly of family Lubomirskiidae (Itskovich et al., 1999), Malawispongiidae are found as polyphyletic assemblage, defined by negative characters and confinement to ancient lakes (Erpenbeck et al., 2019, 2011; Meixner et al., 2007), which supports earlier doubts on a close relationship of current malawispongiid genera (Boury Esnault and Volkmer Ribeiro, 1992). These molecular studies demonstrate ancient lake endemics as independent splits from widespread Spongillidae, indicating that gemmule formation has been lost independently several times in freshwater sponge evolution. However, support for such an hypothesis depends on correctly identified material, preferably from holotypes. In a recent pilot study applying short (=minimalist) barcodes on holotypes of Malawispongia Brien, Spinospongilla Brien and other African freshwater sponges, a close relationship of these malawispongiids could be rejected, demonstrating an independent origin of ancient lake sponge endemism (Erpenbeck et al., 2019). In parallel, initial analyses on non-type material displayed a close relationship of Cortispongilla and Ochridaspongia to Ephydatia fluviatilis Linnaeus, indicating that they might constitute E. fluviatilis ecomorphs with divergent gemmule formation patterns (Itskovich et al., 2013). In the following, we investigate support for such an ‘‘Ephydatia fluviatilis ecomorph” complex using type material and recent material collected in the course of ancient lake biodiversity studies. This includes material from South Africa, Armenia, Tanzania, Lake Ohrid and (for the first time) Lake Prespa, Lake Dojran, and Lake Titicaca. We aim to understand the extent of a widespread sponge species as founder of endemic lineages and resulting implications for freshwater sponge classification.
Material and methods Freshwater sponge tissue derived from historic and recent collections including holotype and cotype material from museums (see Table 1), as well as material collected in the course of sampling campaigns SCOPSCO (Balkans), RiftLink (Africa) and others in South America and the Caucasus, which are deposited in the University of Giessen Systematics and Biodiversity collection (UGSB, see Electronic Supplementary Material (ESM) Table S1; Fig. 1). Institutional acronyms used in this paper are: BMNH, The Natural History Museum, London, United Kingdom; MNH LBIH Natural History Museum Paris, France; MRAC, Musée Royal de l’Afrique Centrale de Tervuren KMMA, Belgium; FW-POR, R. Manconi & R. Pronzato Freshwater Porifera collection, Italy; SNSB-BSPG, The
Table 1 List of historic and reference sponge material examined in the present study. Species
Museum specimen No.
Locality
-type
Ochridaspongia rotunda Arndt, 1937 Ochridaspongia rotunda Arndt, 1937 Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Nudospongilla aster Annandale, 1918 Nudospongilla mappa Annandale, 1918 Nudospongilla reversa Annandale, 1918
ZMB N° 9337
Lake Ohrid
Holo-
FW-POR 695
Lake Ohrid
LBIH DT 3302
Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias
BMNH 13.7.31.4c BMNH 13.7.31.5 BMNH 10.1.1.480 BMNH 14.11.24.24 BMNH 25.11.12.591 BMNH 13.7.31.3 ZMB 4729 ZMB 4728
Holo-
Co– Co– Co–
Bavarian Natural History Collections–Bavarian State Collection for Palaeontology and Geology; UGSB, University of Giessen Systematics and Biodiversity collection, Giessen, Germany; ZMB, Zoologisches Museum für Naturkunde, Humboldt Universität, Berlin, Germany. DNA was extracted using the NucleoSpinÒ Tissue DNA extraction Kit (Macherey-Nagel) following the manufacturer’s protocol. Amplification of the entire ITS region was attempted for all specimens using the primers ITS-RA2-fwd (50 -GTC CCT GCC CTT TGT ACA CA-30 ) in combination with ITS2.2-rvse (50 -CCT GGT TAG TTT CTT TTC CTC CGC-30 ) (Wörheide, 1998) with 45 °C annealing temperature and 1 min elongation time. In case of amplification failure (due to fragmented DNA as a result of long-time storage), amplification of two minimalist ITS-barcoding regions was attempted following Erpenbeck et al. (2019) with the primers: 5.8_Freshies_1180_9f: 50 -GCA CGT CTG TCT GAG CGT CCG-30 (5.8S forward) in combination with ITS2_Freshies_1174_3r: 50 -GCT TCG CAC TTS AAG GGA CGC-30 (ITS2 reverse); and ITS2_Freshies_1176_5f: 50 -TTG CGC GTC GGG AAC TCG AC-30 (ITS2 forward) in combination with 28S_Freshies_1178_7r: 50 -GCT TAT TGA TAT GCT TAA ATT CAG C-30 (28S reverse) with 52 °C and 55 °C annealing temperature respectively with 45 sec elongation time. PCR products were purified with a Freeze-Squeeze (Thuring et al., 1975) and both strands sequenced on an ABI 3730 automated sequencer after cycle sequencing with BigDye-Terminator Mix v3.1 (Applied Biosystems). Sequences were basecalled, trimmed, assembled and checked in CodonCode Aligner v 3.7.1.1 (www.codoncode.com). Spongillid origin of samples was checked with BLAST against NCBI Genbank (www.ncbi.nlm.nih.gov/genbank). Sequences are deposited in the European Nucleotide Archive (www.ebi.ac.uk/ena) under accession numbers LR732049– LR732068 and in the Sponge Barcoding Database (SBD, www. spongebarcoding.org). Sequences were aligned with other available freshwater sponge sequences as published in NCBI Genbank (www. ncbi.nlm.nih.gov) using MAFFT (Katoh and Standley, 2013). Intragenomic polymorphic positions known for Ephydatia fluviatilis (Karlep et al., 2013), were omitted from analyses. Maximum likelihood phylogenetic reconstructions were performed using RAxML 8 (Stamatakis, 2014) as implemented in Geneious 8.1.9 (Kearse et al., 2012) with the GTRGAMMAI model for nucleotide substitution and 100 rapid bootstrap replicates, in parallel with FastTree 2.1.5
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Lake Aygerlich Lake Ohrid Lake Prespa Lake Dojran
Lake Kinneret / Tiberias
Lake Victoria
Lake Titicaca
Grahamstown
Fig. 1. Map of collection locations of the newly analyzed Spongillida samples in this study.
(Price et al., 2010) as implemented in Geneious under the GTR model and Gamma20 likelihood model optimization. Results and discussion Several historic specimens were successfully amplified with the spongillid miniature barcodes, while amplification of full-length ITS failed for this material. The successful 5.8-ITS2 and/or ITS228S miniature barcode amplifications of historic (type-) material of Cortispongilla barroisi (Topsent) and Nudospongilla aster Annandale from the collections of Topsent, Annandale, Dendy and Norman (i.e., collected more than 100 years ago), underline the feasibility of miniature barcodes (Cárdenas and Moore, 2017; Erpenbeck et al., 2019). The holotype of Ochridaspongia rotunda Arndt (ZMB 9337), however, was fixated in formol in the past, which prevents the application of most sequencing strategies on this specimen. The resulting data sets comprised 183 taxa and 196 characters (5.8-ITS2, Fig. 2), respective 189 taxa and 263 characters (ITS2-28S, ESM Fig. S1). The phylogenetic reconstructions (Fig. 2 and ESM Fig. S1) do not recover Spongillidae (Ephydatia, Eunapius, Heterorotula Penney & Racek, Nudospongilla Annandale, and Spongilla) as monophyletic. This corroborates all earlier molecular studies that suggested Spongillidae paraphyly (e.g., Addis and Peterson, 2005; Erpenbeck et al., 2019; Itskovich et al., 2013, 2008) and founder function for most endemic freshwater sponges (Erpenbeck et al., 2011; Meixner et al., 2007). The largest clade is predominantly comprised of Ephydatia fluviatilis and in the following referred to as ‘‘E. fluviatilis group” (Fig. 1 and ESM Fig. S1). Our ITS phylogeny combines Ephydatia fluviatilis and Cortispongilla barroisi, here represented by Topsent’s (1892) holotype (LBIH DT 3302, collected 1890 by Barrois) and samples from Annandale, Dendy and Norman historic collections. The phylogenetic placement of the Cortispongilla specimens is clearly distant from other Malawispongiidae in this analysis, which include the type genera Malawispongia and Spinospongilla (both represented by the holotypes of their respective type species M. echinoides Brien and S. polli Brien). Consequently, holotype material from 60% of all malawispongiid type species suggest polyphyly of this
family. This corroborates earlier studies (Erpenbeck et al., 2019; Itskovich et al., 2013; Meixner et al., 2007) with type material and prompts for abandonment of this family. Their possible new taxonomic re-allocation requires a major revision of Spongillida and is not in the scope of this paper. Absence of gemmule production, regarded as combining morphotrait of the Malawispongiidae, appears to be acquired convergently (see Manconi et al., 1999). Cortispongilla barroisi type material confirms the close relationship to the widespread (and gemmule producing) Ephydatia fluviatilis, as earlier suggested by Itskovich et al. (2013). In their study, Itskovich and coworkers estimated the level of intraspecific variability between Cortispongilla barroisi and Ephydatia syriaca, a further Lake Kinneret/Tiberias species initially described as variety of E. fluviatilis, as not exceeding the level of E. fluviatilis intraspecific variability, and suggested their conspecificity (Itskovich et al., 2013). The historic material sequenced by us, however, displays a different genotype to the C. barroisi sequences currently published in NCBI Genbank. The published sequences lack a 10 bp region at the 30 end of ITS2 forming a perfect hairpin structure after transcription into RNA, distinguishing these clearly from the historic material (see ITS2-28S tree in the ESM Fig. S1). Absence, presence and shape of hairpins can be distinguishing markers in sponge classification (e.g., Voigt et al., 2008), and its absence in E. syriaca would constitute a discriminating character to E. fluviatilis. However, absence of this region is also found in all specimens sequenced from Lake Dojran; they share an ITS genotype from Estonian E. fluviatilis (see Karlep et al., 2013) and also lack this same hairpin region, suggesting an independent loss. ITS sequences of Nudospongilla aster Annandale (co-type BMNH 13.7.31.3) are identical to C. barroisi. Nudospongilla aster, N. mappa Annandale and N. reversa Annandale are endemic to Lake Kinneret and were classified as Nudospongilla by Annandale (1918) due to predominantly encrusting growth form and occurrence in deeper habitats; however, Racek (1974) recognized the three species as C. barroisi ecomorphs (Itskovich et al., 2013; see discussions in Pollingher et al., 1978). His synonymization of N. aster as C. barroisi is corroborated by our molecular results. Sequences from N. mappa and N. reversa holotypes (ZMB 4729 and 4728) were attempted, but could yet not be retrieved.
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Furthermore, Ochridaspongia rotunda, type species of Ochridaspongia, is part of the ‘‘E. fluviatilis group” (Fig. 1), consistent with earlier findings based on CO1 sequence analyses (Meixner et al., 2007). All presently analysed specimens collected from Lake Ohrid form a monophyletic group and are distinct from of Lake Prespa samples, which were analyzed for the first time with molecular methods. Lakes Ohrid and Prespa form ancient sister lakes in the Balkan Peninsula as part of an the Dessaretes European lake group (see overview in Albrecht et al., 2008). Both lakes are connected by karstic cavities (Amataj et al., 2007) and are situated at different altitudes, therefore, about 20% of the lower Lake Ohrid’s water is derived from the higher Lake Prespa (Matzinger et al., 2006). Interconnection between sister lakes can facilitate faunal exchange, or at least establish endemic sister lineages as observed for mollusks in Lakes Ohrid and Prespa (Albrecht et al., 2008; Schultheiß et al., 2008). Our data did not reveal shared or sister lineages for sponges so far. Lake Titicaca sponges were analysed in this study for the first time. Balliviaspongia wirrmanni Boury Esnault & Volkmer-Ribeiro is the only sponge species known from Lake Titicaca (Carney, 1997; Van Soest et al., 2019), likewise member of the ‘‘E. fluviatilis group”. Boury Esnault and Volkmer Ribeiro (1992) already remarked Balliviaspongia Boury Esnault & Volkmer-Ribeiro similarity to E. fluviatilis in terms of choanocyte chamber volume and choanocyte numbers. Balliviaspongia was regarded as Spongillida Incertae Sedis (Manconi and Pronzato, 2002), due to their ‘‘high possibility of convergence as occur in other taxa of ancient lakes” (Manconi et al., 1999). However, our results now strongly suggest that abovementioned putative morphological convergences more likely constitute homologous morphotraits among the ‘‘E. fluviatilis group”. Their endemic genera (Cortispongilla, Balliviaspongia, Ochridaspongia) and cosmopolitan species (E. fluviatilis) display taxonomically relevant similarities when gemmule production is disregarded: they possess oxeas as only (non-gemmular) spicule type, ranging from slender to stout, straight to slightly curved, smooth to spined with acerate tips in overlapping size ranges and also an anisotropic reticulate choanosomal skeleton with paucispicular secondary tracts; scarce spongin, and (with exception of Balliviaspongia) no differentiated ectosomal skeleton (Manconi et al., 1999; Manconi and Pronzato, 2002). The growth form, spicular complement and skeleton organization of B. wirrmanni partly matches with that of Ephydatia spp., as a consequence the former could be considered an eco-morph variety of a South American Ephydatia species (E. fluviatilis is absent in South America). Remaining (minor) differences are reflected in the genetic distinction or were subject to environmental plasticity. For example, some aspects of spicular complement, skeletal architecture, and growth form are diverging from E. fluviatilis; that notwithstanding, it could be possible that O. rotunda descends from an ancestor shared with E. fluviatilis that
3 Fig. 2. Maximum likelihood reconstruction of the 5.8S-ITS2 miniature barcoding regions. Taxa in boldface are newly sequenced for this study. Sequences of taxa in regular font are taken from Genbank or Erpenbeck et al. (2009). Taxa in blue depict ancient lake (alleged) endemics without gemmule production. Number codes with taxon names are NCBI Genbank accession numbers, or museum collection numbers of the BMNH (BMNHxxxx.xx.xx.xx), SNSB-BSPG (GWxxxxx) and MRAC (MRACxxxx), alternatively voucher numbers of the UGSB (UGSBxxxxx), MNH LBIH (MNH LBIH xxxxxx), or the R. Manconi & R. Pronzato Freshwater Porifera collection (FW-PORxxx). For legibility, bootstrap support >50 is provided at selected branches only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
D. Erpenbeck et al. / Journal of Great Lakes Research xxx (xxxx) xxx
has lost the ability to gemmulate, the smooth oxeas megasclere, and acquired a subspherical growth form with a large, central oscular area at the top. Also, E. fluviatilis and E. syriaca Topsent differ by spicule sizes and texture (with intermediate expression in the Lake Kinneret/Tiberias E. syriaca); likewise, Cortispongilla (including morphs assigned to Nudospongilla) differs from Ephydatia by megasclere thickness (see Itskovich et al., 2013 for overview and discussion). Several experimental studies have now documented the variability of spicule size and shape based on environmental abiotic factors in megascleres (Cárdenas and Rapp, 2013; Maldonado et al., 1999), as well as gemmuloscleres, which has extensively been studied for E. fluviatilis (Poirrier, 1974). Consequently, the validity of species in the ‘‘E. fluviatilis group” requires critical re-evaluation (Itskovich et al., 2013). Structural morphotraits of the freshwater sponge lineages of the ‘‘E. fluviatilis group” do not contradict the molecular phylogenetic results. They underline the evolution of endemic, gemmulelacking lineages in ancient lake habitats (e.g., Ochridaspongia in Lake Ohrid, Cortispongilla in Lake Kinneret/Tiberias, Balliviaspongia in Lake Titicaca) from a gemmule-bearing founder lineage closely related to (or identical with) E. fluviatilis. The loss of gemmule production and asexual reproduction correlates with the ecological condition of the lakes (Brien and Govaert-Mallebrancke, 1958), which experience less dramatic changes in comparison to other freshwater habitats (see discussion in Boury Esnault and Volkmer Ribeiro, 1992; Manconi and Pronzato, 2016, 2002). Upcoming genomic studies will reveal the underlying mechanisms of asexual reproduction in sponges, which is integral for radiation and dispersal. Furthermore, fossil data imply a widespread radiation of Ephydatia already in the Miocene (see review in Pronzato et al., 2017). The Neotropical Biorealm, which includes Lake Titicaca, hosted E. chileana Pisera & Saez (Late Miocene, Chile) while the Palaearctic Biorealm, which includes Lake Ohrid and Lake Kinneret/Tiberias, hosted E. fossilis Traxler (Miocene) and E. gutenbergiana Müller, Zahn & Maidhoff (Late Eocene) (see Pisera et al., 2016). Likewise, Pisera et al. (2019) report Ochridaspongia spicule fossils from middle Miocene lacustrine deposits in the central Bosnia and Herzegovina, concluding that Ochridaspongia origins are not in Lake Ohrid but earlier in the Dinarides Lake System during the middle Miocene alongside Ephydatia. In conclusion, our results now prompt for a reclassification of freshwater sponges, in particular in respect to the polyphyly of Spongillidae as the largest and most widespread distributed freshwater sponge taxon. Similar to Ephydatia fluviatilis, E. muelleri, Eunapius spp., and Spongilla spp., could constitute unrelated gemmular-bearing founder groups for diverging, spatially more restricted or even endemic lineages (Figs. 1 and 2) and reference points for a new classification. The traditional morphology-based classification concept of freshwater sponges needs re-appraisal and especially the paraphyly of well-established taxa (e.g., Ephydatia fluviatilis and E. muelleri) needs full consideration in an integrative taxonomic approach to help in resolving still uncertain phylogenetic relationships of the order Spongillida.
Acknowledgements We thank the Deutsche Forschungsgemeinschaft (DFG: Al1076/9), SYNTHESYS (BE-TAF 2601), and Lehre@LMU for financial support to this study. Gabriele Büttner und Simone Schätzle are thanked for various aspects of the molecular work. GW acknowledges funding through the LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative. RM acknowledges financial support from Fondazione Sardegna (FS-2016). We thank T. Hauffe, S. Sereda, C.
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Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages Dirk Erpenbeck a,b,⇑, Adrian Galitz a, Gert Wörheide a,b,c, Christian Albrecht d, Roberto Pronzato f, Renata Manconi e,f,⇑ a
Department of Earth & Environmental Sciences, Palaeontology and Geobiology, Ludwig-Maximilians-Universität München, Munich, Germany GeoBio-CenterLMU, Ludwig-Maximilians-Universität München, Munich, Germany SNSB – Bayerische Staatssammlung für Paläontologie und Geologie, Munich, Germany d Department of Animal Ecology and Systematics, Systematics and Biodiversity Group, Justus Liebig University Giessen, Giessen, Germany e ` di Genova, Genova, Italy Dipartimento di Scienze della Terra, dell’Ambiente e della Vita, Universita f Dipartimento Medicina Veterinaria, Università di Sassari, Sassari, Italy b c
a r t i c l e
i n f o
Article history: Received 3 May 2019 Accepted 16 September 2019 Available online xxxx Communicated by: Bjorn Stelbrink
Keywords: Freshwater sponges Spongillida Ancient lakes Ochridaspongia Cortispongilla Balliviaspongia
a b s t r a c t Spongillida (freshwater sponges) constitute an integral part of the world’s lacustrine ecosystems. Their radiation and worldwide distribution into nearly every freshwater habitat is predominantly facilitated by the formation of so-called gemmules, i.e., globular endurance and propagation stages. Widespread species produce gemmules in a wide array of morphs, while in (ancient) lake endemic lineages gemmules are absent. Contrary to current classification, earlier molecular studies on non-type material indicated that gemmule-lacking lineages are unrelated, and endemic taxa were derived from a cosmopolitan (and paraphyletic) founder lineage. In this study we investigate this hypothesis with type material, particularly focussing on endemic taxa potentially derived from common ancestor shared with the widespread Ephydatia fluviatilis. We demonstrate that taxa regarded as endemic from Lake Ohrid (Ochridaspongia), Lake Kinneret/Tiberias (Cortispongilla) and Lake Titicaca (Balliviaspongia) are derived from the founder lineage, but unrelated to other, particularly Afrotropical, ancient lake sponges, and discuss implications for understanding freshwater sponge phylogeny and evolution. Ó 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Introduction Freshwater sponges (Porifera: Demospongiae: Spongillida) are pivotal organisms in freshwater systems on all continents except Antarctica (Manconi and Pronzato, 2008). They have colonized almost every limnic habitat from lentic mountain lakes to brackish lotic estuaries (Manconi and Pronzato, 2007), from abyssal depths of ancient lakes (Crane et al., 1991; Itskovich et al., 2015) to surface regions, and above as ‘‘arboreal sponges” (see Manconi and Pronzato, 2016 for overview). Present-day freshwater sponges (order Spongillida Manconi & Pronzato) constitute a monophyletic group (e.g., Itskovich et al., 1999), implying that successful transition from the marine to brackish and freshwater habitats occurred
⇑ Corresponding authors at: Department of Earth & Environmental Sciences, Palaeontology and Geobiology, Ludwig-Maximilians-Universität München, Munich, Germany (D. Erpenbeck). Dipartimento Medicina Veterinaria, Università di Sassari, Sassari, Italy (R. Manconi). E-mail addresses: [email protected] (D. Erpenbeck), [email protected] (R. Manconi).
only once. Transition was followed by extensive dispersal and radiation to presently ~240 species (51 genera, Manconi and Pronzato, 2015; Pronzato et al., 2017), which exceeds numbers of other sessile freshwater filter feeders like Cnidaria and Bryozoa (Manconi and Pronzato, 2007). Key adaptive traits facilitating spongillid dispersal and radiation are high plasticity of body plan, physiology, life cycle and reproductive strategies, but particularly cryptobiosis by gemmules (Manconi and Pronzato, 2007). Gemmules constitute globular dormant resting bodies that may endure unfavourable abiotic conditions, including drought and freezing, which affect freshwater systems more frequently than marine (Frost et al., 1982; Pronzato and Manconi, 1995, 1994) Gemmules also facilitate easy dispersal as they can possess ‘‘pneumatic layers” for floating or potentially wind-driven dispersal, and/or possess a spiny outer theca to hook onto carriers such as birds (see overview in Manconi and Pronzato, 2007). Consequently, presence or absence of gemmules and differences in gemmular morphology is believed to be a key for dispersal success of spongillid lineages, facilitating nearly global distribution of the genera Spongilla Lamarck or (to a lesser extent) Ephydatia Lamouroux and Eunapius Gray (all family
https://doi.org/10.1016/j.jglr.2019.09.028 0380-1330/Ó 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Spongillidae Gray). Several other genera, however, apparently lack gemmule formation and remain strictly endemic to small spatial ranges (e.g., Cortispongilla Annandale to Lake Kinneret/Tiberias and River Jordan) (Manconi and Pronzato, 2002). Ancient lakes (here defined as lakes older than 130,000 years, i.e., existing for at least a glacial cycle, Hampton et al., 2018) are renownded for their invertebrate radiations (e.g., Genner et al., 2007) and harbour a wide range of freshwater sponge lineages. These include gemmule-lacking endemic taxa like Lubomirskiidae Weltner (Lake Baikal, four genera), and Metschnikowiidae Czerniavsky (Caspian Sea, monogeneric), but also Ochridaspongia Arndt (Lake Ohrid), Pachydictyum Weltner (Lake Poso), Spinospongilla Brien (Lake Tanganyika) and Malawispongia Brien (Lake Malawi) (Manconi and Pronzato, 2002). The latter genera are combined with Cortispongilla into the family Malawispongiidae Manconi and Pronzato, which was firstly erected as Globulospongillinae by Brien (1973) and Globulospongillidae by Racek (1974) to accommodate gemmule-lacking ancient lake taxa and now comprises five endemic, predominantly monotypic genera with a notably disjunct distribution (Manconi and Pronzato, 2002). While molecular data support the monophyly of family Lubomirskiidae (Itskovich et al., 1999), Malawispongiidae are found as polyphyletic assemblage, defined by negative characters and confinement to ancient lakes (Erpenbeck et al., 2019, 2011; Meixner et al., 2007), which supports earlier doubts on a close relationship of current malawispongiid genera (Boury Esnault and Volkmer Ribeiro, 1992). These molecular studies demonstrate ancient lake endemics as independent splits from widespread Spongillidae, indicating that gemmule formation has been lost independently several times in freshwater sponge evolution. However, support for such an hypothesis depends on correctly identified material, preferably from holotypes. In a recent pilot study applying short (=minimalist) barcodes on holotypes of Malawispongia Brien, Spinospongilla Brien and other African freshwater sponges, a close relationship of these malawispongiids could be rejected, demonstrating an independent origin of ancient lake sponge endemism (Erpenbeck et al., 2019). In parallel, initial analyses on non-type material displayed a close relationship of Cortispongilla and Ochridaspongia to Ephydatia fluviatilis Linnaeus, indicating that they might constitute E. fluviatilis ecomorphs with divergent gemmule formation patterns (Itskovich et al., 2013). In the following, we investigate support for such an ‘‘Ephydatia fluviatilis ecomorph” complex using type material and recent material collected in the course of ancient lake biodiversity studies. This includes material from South Africa, Armenia, Tanzania, Lake Ohrid and (for the first time) Lake Prespa, Lake Dojran, and Lake Titicaca. We aim to understand the extent of a widespread sponge species as founder of endemic lineages and resulting implications for freshwater sponge classification.
Material and methods Freshwater sponge tissue derived from historic and recent collections including holotype and cotype material from museums (see Table 1), as well as material collected in the course of sampling campaigns SCOPSCO (Balkans), RiftLink (Africa) and others in South America and the Caucasus, which are deposited in the University of Giessen Systematics and Biodiversity collection (UGSB, see Electronic Supplementary Material (ESM) Table S1; Fig. 1). Institutional acronyms used in this paper are: BMNH, The Natural History Museum, London, United Kingdom; MNH LBIH Natural History Museum Paris, France; MRAC, Musée Royal de l’Afrique Centrale de Tervuren KMMA, Belgium; FW-POR, R. Manconi & R. Pronzato Freshwater Porifera collection, Italy; SNSB-BSPG, The
Table 1 List of historic and reference sponge material examined in the present study. Species
Museum specimen No.
Locality
-type
Ochridaspongia rotunda Arndt, 1937 Ochridaspongia rotunda Arndt, 1937 Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Cortispongilla barroisi (Topsent, 1892) Nudospongilla aster Annandale, 1918 Nudospongilla mappa Annandale, 1918 Nudospongilla reversa Annandale, 1918
ZMB N° 9337
Lake Ohrid
Holo-
FW-POR 695
Lake Ohrid
LBIH DT 3302
Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias Lake Kinneret/ Tiberias
BMNH 13.7.31.4c BMNH 13.7.31.5 BMNH 10.1.1.480 BMNH 14.11.24.24 BMNH 25.11.12.591 BMNH 13.7.31.3 ZMB 4729 ZMB 4728
Holo-
Co– Co– Co–
Bavarian Natural History Collections–Bavarian State Collection for Palaeontology and Geology; UGSB, University of Giessen Systematics and Biodiversity collection, Giessen, Germany; ZMB, Zoologisches Museum für Naturkunde, Humboldt Universität, Berlin, Germany. DNA was extracted using the NucleoSpinÒ Tissue DNA extraction Kit (Macherey-Nagel) following the manufacturer’s protocol. Amplification of the entire ITS region was attempted for all specimens using the primers ITS-RA2-fwd (50 -GTC CCT GCC CTT TGT ACA CA-30 ) in combination with ITS2.2-rvse (50 -CCT GGT TAG TTT CTT TTC CTC CGC-30 ) (Wörheide, 1998) with 45 °C annealing temperature and 1 min elongation time. In case of amplification failure (due to fragmented DNA as a result of long-time storage), amplification of two minimalist ITS-barcoding regions was attempted following Erpenbeck et al. (2019) with the primers: 5.8_Freshies_1180_9f: 50 -GCA CGT CTG TCT GAG CGT CCG-30 (5.8S forward) in combination with ITS2_Freshies_1174_3r: 50 -GCT TCG CAC TTS AAG GGA CGC-30 (ITS2 reverse); and ITS2_Freshies_1176_5f: 50 -TTG CGC GTC GGG AAC TCG AC-30 (ITS2 forward) in combination with 28S_Freshies_1178_7r: 50 -GCT TAT TGA TAT GCT TAA ATT CAG C-30 (28S reverse) with 52 °C and 55 °C annealing temperature respectively with 45 sec elongation time. PCR products were purified with a Freeze-Squeeze (Thuring et al., 1975) and both strands sequenced on an ABI 3730 automated sequencer after cycle sequencing with BigDye-Terminator Mix v3.1 (Applied Biosystems). Sequences were basecalled, trimmed, assembled and checked in CodonCode Aligner v 3.7.1.1 (www.codoncode.com). Spongillid origin of samples was checked with BLAST against NCBI Genbank (www.ncbi.nlm.nih.gov/genbank). Sequences are deposited in the European Nucleotide Archive (www.ebi.ac.uk/ena) under accession numbers LR732049– LR732068 and in the Sponge Barcoding Database (SBD, www. spongebarcoding.org). Sequences were aligned with other available freshwater sponge sequences as published in NCBI Genbank (www. ncbi.nlm.nih.gov) using MAFFT (Katoh and Standley, 2013). Intragenomic polymorphic positions known for Ephydatia fluviatilis (Karlep et al., 2013), were omitted from analyses. Maximum likelihood phylogenetic reconstructions were performed using RAxML 8 (Stamatakis, 2014) as implemented in Geneious 8.1.9 (Kearse et al., 2012) with the GTRGAMMAI model for nucleotide substitution and 100 rapid bootstrap replicates, in parallel with FastTree 2.1.5
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Lake Aygerlich Lake Ohrid Lake Prespa Lake Dojran
Lake Kinneret / Tiberias
Lake Victoria
Lake Titicaca
Grahamstown
Fig. 1. Map of collection locations of the newly analyzed Spongillida samples in this study.
(Price et al., 2010) as implemented in Geneious under the GTR model and Gamma20 likelihood model optimization. Results and discussion Several historic specimens were successfully amplified with the spongillid miniature barcodes, while amplification of full-length ITS failed for this material. The successful 5.8-ITS2 and/or ITS228S miniature barcode amplifications of historic (type-) material of Cortispongilla barroisi (Topsent) and Nudospongilla aster Annandale from the collections of Topsent, Annandale, Dendy and Norman (i.e., collected more than 100 years ago), underline the feasibility of miniature barcodes (Cárdenas and Moore, 2017; Erpenbeck et al., 2019). The holotype of Ochridaspongia rotunda Arndt (ZMB 9337), however, was fixated in formol in the past, which prevents the application of most sequencing strategies on this specimen. The resulting data sets comprised 183 taxa and 196 characters (5.8-ITS2, Fig. 2), respective 189 taxa and 263 characters (ITS2-28S, ESM Fig. S1). The phylogenetic reconstructions (Fig. 2 and ESM Fig. S1) do not recover Spongillidae (Ephydatia, Eunapius, Heterorotula Penney & Racek, Nudospongilla Annandale, and Spongilla) as monophyletic. This corroborates all earlier molecular studies that suggested Spongillidae paraphyly (e.g., Addis and Peterson, 2005; Erpenbeck et al., 2019; Itskovich et al., 2013, 2008) and founder function for most endemic freshwater sponges (Erpenbeck et al., 2011; Meixner et al., 2007). The largest clade is predominantly comprised of Ephydatia fluviatilis and in the following referred to as ‘‘E. fluviatilis group” (Fig. 1 and ESM Fig. S1). Our ITS phylogeny combines Ephydatia fluviatilis and Cortispongilla barroisi, here represented by Topsent’s (1892) holotype (LBIH DT 3302, collected 1890 by Barrois) and samples from Annandale, Dendy and Norman historic collections. The phylogenetic placement of the Cortispongilla specimens is clearly distant from other Malawispongiidae in this analysis, which include the type genera Malawispongia and Spinospongilla (both represented by the holotypes of their respective type species M. echinoides Brien and S. polli Brien). Consequently, holotype material from 60% of all malawispongiid type species suggest polyphyly of this
family. This corroborates earlier studies (Erpenbeck et al., 2019; Itskovich et al., 2013; Meixner et al., 2007) with type material and prompts for abandonment of this family. Their possible new taxonomic re-allocation requires a major revision of Spongillida and is not in the scope of this paper. Absence of gemmule production, regarded as combining morphotrait of the Malawispongiidae, appears to be acquired convergently (see Manconi et al., 1999). Cortispongilla barroisi type material confirms the close relationship to the widespread (and gemmule producing) Ephydatia fluviatilis, as earlier suggested by Itskovich et al. (2013). In their study, Itskovich and coworkers estimated the level of intraspecific variability between Cortispongilla barroisi and Ephydatia syriaca, a further Lake Kinneret/Tiberias species initially described as variety of E. fluviatilis, as not exceeding the level of E. fluviatilis intraspecific variability, and suggested their conspecificity (Itskovich et al., 2013). The historic material sequenced by us, however, displays a different genotype to the C. barroisi sequences currently published in NCBI Genbank. The published sequences lack a 10 bp region at the 30 end of ITS2 forming a perfect hairpin structure after transcription into RNA, distinguishing these clearly from the historic material (see ITS2-28S tree in the ESM Fig. S1). Absence, presence and shape of hairpins can be distinguishing markers in sponge classification (e.g., Voigt et al., 2008), and its absence in E. syriaca would constitute a discriminating character to E. fluviatilis. However, absence of this region is also found in all specimens sequenced from Lake Dojran; they share an ITS genotype from Estonian E. fluviatilis (see Karlep et al., 2013) and also lack this same hairpin region, suggesting an independent loss. ITS sequences of Nudospongilla aster Annandale (co-type BMNH 13.7.31.3) are identical to C. barroisi. Nudospongilla aster, N. mappa Annandale and N. reversa Annandale are endemic to Lake Kinneret and were classified as Nudospongilla by Annandale (1918) due to predominantly encrusting growth form and occurrence in deeper habitats; however, Racek (1974) recognized the three species as C. barroisi ecomorphs (Itskovich et al., 2013; see discussions in Pollingher et al., 1978). His synonymization of N. aster as C. barroisi is corroborated by our molecular results. Sequences from N. mappa and N. reversa holotypes (ZMB 4729 and 4728) were attempted, but could yet not be retrieved.
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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Furthermore, Ochridaspongia rotunda, type species of Ochridaspongia, is part of the ‘‘E. fluviatilis group” (Fig. 1), consistent with earlier findings based on CO1 sequence analyses (Meixner et al., 2007). All presently analysed specimens collected from Lake Ohrid form a monophyletic group and are distinct from of Lake Prespa samples, which were analyzed for the first time with molecular methods. Lakes Ohrid and Prespa form ancient sister lakes in the Balkan Peninsula as part of an the Dessaretes European lake group (see overview in Albrecht et al., 2008). Both lakes are connected by karstic cavities (Amataj et al., 2007) and are situated at different altitudes, therefore, about 20% of the lower Lake Ohrid’s water is derived from the higher Lake Prespa (Matzinger et al., 2006). Interconnection between sister lakes can facilitate faunal exchange, or at least establish endemic sister lineages as observed for mollusks in Lakes Ohrid and Prespa (Albrecht et al., 2008; Schultheiß et al., 2008). Our data did not reveal shared or sister lineages for sponges so far. Lake Titicaca sponges were analysed in this study for the first time. Balliviaspongia wirrmanni Boury Esnault & Volkmer-Ribeiro is the only sponge species known from Lake Titicaca (Carney, 1997; Van Soest et al., 2019), likewise member of the ‘‘E. fluviatilis group”. Boury Esnault and Volkmer Ribeiro (1992) already remarked Balliviaspongia Boury Esnault & Volkmer-Ribeiro similarity to E. fluviatilis in terms of choanocyte chamber volume and choanocyte numbers. Balliviaspongia was regarded as Spongillida Incertae Sedis (Manconi and Pronzato, 2002), due to their ‘‘high possibility of convergence as occur in other taxa of ancient lakes” (Manconi et al., 1999). However, our results now strongly suggest that abovementioned putative morphological convergences more likely constitute homologous morphotraits among the ‘‘E. fluviatilis group”. Their endemic genera (Cortispongilla, Balliviaspongia, Ochridaspongia) and cosmopolitan species (E. fluviatilis) display taxonomically relevant similarities when gemmule production is disregarded: they possess oxeas as only (non-gemmular) spicule type, ranging from slender to stout, straight to slightly curved, smooth to spined with acerate tips in overlapping size ranges and also an anisotropic reticulate choanosomal skeleton with paucispicular secondary tracts; scarce spongin, and (with exception of Balliviaspongia) no differentiated ectosomal skeleton (Manconi et al., 1999; Manconi and Pronzato, 2002). The growth form, spicular complement and skeleton organization of B. wirrmanni partly matches with that of Ephydatia spp., as a consequence the former could be considered an eco-morph variety of a South American Ephydatia species (E. fluviatilis is absent in South America). Remaining (minor) differences are reflected in the genetic distinction or were subject to environmental plasticity. For example, some aspects of spicular complement, skeletal architecture, and growth form are diverging from E. fluviatilis; that notwithstanding, it could be possible that O. rotunda descends from an ancestor shared with E. fluviatilis that
3 Fig. 2. Maximum likelihood reconstruction of the 5.8S-ITS2 miniature barcoding regions. Taxa in boldface are newly sequenced for this study. Sequences of taxa in regular font are taken from Genbank or Erpenbeck et al. (2009). Taxa in blue depict ancient lake (alleged) endemics without gemmule production. Number codes with taxon names are NCBI Genbank accession numbers, or museum collection numbers of the BMNH (BMNHxxxx.xx.xx.xx), SNSB-BSPG (GWxxxxx) and MRAC (MRACxxxx), alternatively voucher numbers of the UGSB (UGSBxxxxx), MNH LBIH (MNH LBIH xxxxxx), or the R. Manconi & R. Pronzato Freshwater Porifera collection (FW-PORxxx). For legibility, bootstrap support >50 is provided at selected branches only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028
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has lost the ability to gemmulate, the smooth oxeas megasclere, and acquired a subspherical growth form with a large, central oscular area at the top. Also, E. fluviatilis and E. syriaca Topsent differ by spicule sizes and texture (with intermediate expression in the Lake Kinneret/Tiberias E. syriaca); likewise, Cortispongilla (including morphs assigned to Nudospongilla) differs from Ephydatia by megasclere thickness (see Itskovich et al., 2013 for overview and discussion). Several experimental studies have now documented the variability of spicule size and shape based on environmental abiotic factors in megascleres (Cárdenas and Rapp, 2013; Maldonado et al., 1999), as well as gemmuloscleres, which has extensively been studied for E. fluviatilis (Poirrier, 1974). Consequently, the validity of species in the ‘‘E. fluviatilis group” requires critical re-evaluation (Itskovich et al., 2013). Structural morphotraits of the freshwater sponge lineages of the ‘‘E. fluviatilis group” do not contradict the molecular phylogenetic results. They underline the evolution of endemic, gemmulelacking lineages in ancient lake habitats (e.g., Ochridaspongia in Lake Ohrid, Cortispongilla in Lake Kinneret/Tiberias, Balliviaspongia in Lake Titicaca) from a gemmule-bearing founder lineage closely related to (or identical with) E. fluviatilis. The loss of gemmule production and asexual reproduction correlates with the ecological condition of the lakes (Brien and Govaert-Mallebrancke, 1958), which experience less dramatic changes in comparison to other freshwater habitats (see discussion in Boury Esnault and Volkmer Ribeiro, 1992; Manconi and Pronzato, 2016, 2002). Upcoming genomic studies will reveal the underlying mechanisms of asexual reproduction in sponges, which is integral for radiation and dispersal. Furthermore, fossil data imply a widespread radiation of Ephydatia already in the Miocene (see review in Pronzato et al., 2017). The Neotropical Biorealm, which includes Lake Titicaca, hosted E. chileana Pisera & Saez (Late Miocene, Chile) while the Palaearctic Biorealm, which includes Lake Ohrid and Lake Kinneret/Tiberias, hosted E. fossilis Traxler (Miocene) and E. gutenbergiana Müller, Zahn & Maidhoff (Late Eocene) (see Pisera et al., 2016). Likewise, Pisera et al. (2019) report Ochridaspongia spicule fossils from middle Miocene lacustrine deposits in the central Bosnia and Herzegovina, concluding that Ochridaspongia origins are not in Lake Ohrid but earlier in the Dinarides Lake System during the middle Miocene alongside Ephydatia. In conclusion, our results now prompt for a reclassification of freshwater sponges, in particular in respect to the polyphyly of Spongillidae as the largest and most widespread distributed freshwater sponge taxon. Similar to Ephydatia fluviatilis, E. muelleri, Eunapius spp., and Spongilla spp., could constitute unrelated gemmular-bearing founder groups for diverging, spatially more restricted or even endemic lineages (Figs. 1 and 2) and reference points for a new classification. The traditional morphology-based classification concept of freshwater sponges needs re-appraisal and especially the paraphyly of well-established taxa (e.g., Ephydatia fluviatilis and E. muelleri) needs full consideration in an integrative taxonomic approach to help in resolving still uncertain phylogenetic relationships of the order Spongillida.
Acknowledgements We thank the Deutsche Forschungsgemeinschaft (DFG: Al1076/9), SYNTHESYS (BE-TAF 2601), and Lehre@LMU for financial support to this study. Gabriele Büttner und Simone Schätzle are thanked for various aspects of the molecular work. GW acknowledges funding through the LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative. RM acknowledges financial support from Fondazione Sardegna (FS-2016). We thank T. Hauffe, S. Sereda, C.
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Please cite this article as: D. Erpenbeck, A. Galitz, G. Wörheide et al., Having the balls to colonize – The Ephydatia fluviatilis group and the origin of (ancient) lake ‘‘endemic” sponge lineages, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.09.028