Introduction

Seagrass symbionts represent a very heterogeneous group of epi- and endophytic organisms differing in size from macro- to microscopic species. They occur in all possible niches including the phyllosphere, the rhizoplane, and the inside of a seagrass body (either inter- or intracellularly) and perform various functions along the parasitism–mutualism continuum [1]. In a simplified way, seagrass microbial symbionts can be typically divided into bacteria [2,3,4,5], fungi [6,7,8,9], and protists [10,11,12] that are, perhaps except for Labyrinthula zosterae (Labyrinthulomycetes), the putative agent of eelgrass wasting disease [13, 14], considerably understudied.

Among many other taxonomic/ecological groups, seagrass protist symbionts include phytomyxids (SAR: Cercozoa: Phytomyxea), sometimes traditionally referred to as “plasmodiophorids,” an obscure and often neglected clade of biotrophic intracellular symbionts, which, within the marine ecosystem, also colonize brown algae and diatoms [15]. In contrast to their terrestrial counterparts which often have a grave impact on crop plants [16], very little is known about marine species, despite their supposed significant ecological roles in marine ecosystems [10]. Nevertheless, similarly to the terrestrial species, they are also treated as pathogens [17], although processes connected with their infection are poorly understood and for some species, it is unclear how or if at all they influence the fitness of the seagrass [10]. Finally, the reported incidence of seagrass-parasitizing phytomyxids is in general low, rather fragmentary and with driving factors mostly unknown [18,19,20].

There are four autochthonous seagrass species placed in three genera (Cymodocea, Posidonia, and Zostera) in the Mediterranean Sea, and only a few reports on their colonization by phytomyxids are available. Interestingly, these are limited to Plasmodiophora bicaudata infecting Zostera noltii at a single locality in southern France [21, 22] and another one in southwestern Croatia [19]—the most recent report being from 1971 [19]. The Mediterranean Sea is also home to the alien seagrass Halophila stipulacea, one of the first Lessepsian migrants to cross the Suez Canal on its way from the Indo-Pacific [23], and to our knowledge, there are only two published reports on its colonization with phytomyxids in the Mediterranean: one is from the Strait of Messina (Sicily, southern Italy) [24] and one from the Aegean coast of Turkey [25]. This scarcity contrasts with the already large and still expanding distribution area of H. stipulacea in the Mediterranean [26, 27]. While P. bicaudata has been studied and described quite in detail [22, 28] and is considered to be a specific parasite of the seagrass genus Zostera [19], the identity of the phytomyxid infecting H. stipulacea in the Mediterranean Sea is far less clear.

Arguably the first phytomyxid reported to infect Halophila was described more than 100 years ago as Plasmodiophora halophilae on Halophila ovalis from today’s Nusa Kambangan, Java, Indonesia [29], but has never been reported again. As a result, some authors consider it a doubtful species [30, 31]. The symbiont of H. stipulacea in the Mediterranean, firstly reported by Marziano et al. in 1995 [24], seems to be related to the one of H. ovalis from Java. Indeed, Marziano et al. [24] at first “considered the observed parasite to be P. halophilae” (p. 165 in [24]); however, after “further observations on Italian material” (p. 165 in [24]), they decided to reassign the phytomyxid to Tetramyxa parasitica. This decision was based mostly on the observation of spores arranged in tetrads (11% of the observed spore arrangements). However, similarly to the report from the Aegean coast of Turkey [25], most of the spores were actually arranged in dyads (60%) followed by single spores (20%) [24]. Tetramyxa parasitica was originally described by Goebel in 1884 [32] from the salt-tolerant aquatic plant Ruppia rostellata in Northern Germany. In Europe, it has been time by time found in the Baltic Sea and the North Sea on Ruppia and another salt-tolerant aquatic genus Zannichellia [33, 34] but never on seagrasses in the comparably saltier Mediterranean Sea. Most recently, in 2017, a symbiosis morphologically similar to that described by Marziano et al. [24] was reported on the same host at one site in the Aegean Sea in Turkey and the respective phytomyxid was assigned as Plasmodiophora cf. halophilae [25]. Indeed, since H. stipulacea is originally from the Indo-Pacific region and phytomyxids are obligate biotrophs, it is plausible that both the host and its parasite co-migrated along the Lessepsian route together, suggesting a close relationship of the Mediterranean phytomyxid with P. halophilae [25, 35].

To investigate the relationship of the phytomyxid symbiosis recently found in Turkey [25] with that previously reported in Sicily [24] as well as to better understand on-site ecology of the respective phytomyxid(s), four sampling campaigns to Southern Italy were organized in search for the characteristic galls on H. stipulacea. Upon re-discovering the symbiosis in Sicily, we also focused on screening of its development dynamics, colonization levels, possible dispersal modes, and its phylogenetic placement based on the analyses of the 18S rRNA gene.

Materials and Methods

Herbarium Material Examinations

To decide about the relationship between P. halophilae on H. ovalis from Java as reported by Ferdinandsen and Winge [29] and “T. parasitica” on H. stipulacea from Sicily as reported by Marziano et al. [24], a series of attempts have been made to obtain herbarium material from the Botanical Museum in Copenhagen, Denmark, and the Herbarium of the University of Messina, respectively. Unfortunately, it was impossible to get these specimens on loan as they were unavailable and most probably had been lost (also see [25]). On the other hand, permanent slides with thin transverse sections of the H. stipulacea galls investigated by Marziano et al. [24] were obtained from the Herbarium in Messina and examined, using light microscopy. Additionally, a few micrographs of phytomyxid spores originating from H. stipulacea galls collected by chance at a site in northeast Sicily in around 2015 (Tono (Casabianca); Table 1) by one of us (G. M. G.) were also examined.

Table 1 List of the localities investigated in this study during four sampling campaigns (2015–2018)

Sampling

Four sampling campaigns were carried out during 3 years (December 2015, September 2017, May/June 2018, and September 2018) in southern Italy at 16 different localities (Fig. 1). Eight of them were chosen based on literature searches, i.e., papers reporting Halophila stipulacea occurrence [24, 26, 36, 37] and personal communications with local scuba dive operators/scientists; the remaining eight were chosen randomly (Table 1).

Fig. 1
figure 1

Localities investigated in this study. a Study sites were located in the transition zone between the Western and the Eastern Basin of the Mediterranean Sea (black rectangle). b Detailed locations of the investigated sites in the Strait of Messina and its surroundings (dots). Scale bar = 100 km. Original maps were downloaded from the USGS National Map Viewer (public domain at http://viewer.nationalmap.gov/viewer/) for a and the Maps at the CIA (public domain at https://www.cia.gov/library/publications/the-world-factbook/index.html) for b. This figure is similar but not identical to the original images and is therefore for illustrative purposes only

Seagrass samples were collected using scuba diving, the collection depth and average water temperature were measured with a diving computer (Freedom, Divesoft, Czech Republic; Vytec or Vyper Novo, Suunto, Finland), seawater salinity was measured during the 2018 campaigns using a portable optical refractometer for seawater RSA1-ATC (www.refraktometr.cz, Czech Republic), and in situ photo-documentation was done with a Canon G10 camera in a WP-DC28 underwater case. Collected samples were stored in plastic tubes filled with seawater which was upon surfacing substituted with 30% ethanol in seawater in ca. half of the samples. Samples were then transported to the laboratory for further examination; representative specimens were washed with tap water and transferred to a 70% ethanol solution in deionized water or dried at room temperature and then deposited in the Herbarium of the Institute of Botany, Czech Academy of Sciences, Průhonice (PRA).

For measurement of the intensity of the phytomyxid colonization (found only at one locality), three microsites with the presence of the symbiosis were randomly chosen in 2017 and 2018 and at each of them, one plastic box for a diving mask (approx. volume 1.5 l) was filled with the seagrass biomass (leaves + rhizomes + roots). Upon surfacing, all collected leaves were checked for the presence of the symbiosis and according to gall coloration assigned into the three following categories: (i) the early developmental stage containing sporogenic plasmodia (referred to as the “whitish” stage in [25]), (ii) later developmental stages containing cleaved plasmodia and fully developed resting spores (referred to as the “yellowish” and “blackish” developmental stages in [25], respectively), and (iii) no colonization.

Stereomicroscopy and Light and Scanning Electron Microscopy

Firstly, randomly selected rhizomes with leaves as well as detached leaves from the 2018 collection (see “Results”) were screened with an Olympus SZX12 stereomicroscope. Secondly, paraffin thin sections from both infected and non-infected petioles were prepared as detailed in [38]. These sections were eventually mounted into permanent slides and screened at high magnifications (× 400–× 1000) with an Olympus BX60 upright microscope equipped with differential interference contrast. Micrographs were taken with an Olympus DP70 camera using the QuickPHOTO MICRO 2.3 software (Promicra Ltd., Czech Republic); the Deep Focus Mode was employed when needed (Promicra). Thirdly, scanning electron microscopy (SEM) of semi-thin hand sections was performed using a FEI Quanta 200 scanning electron microscope in the ESEM mode at low temperatures (− 6 to − 3 °C). Micrographs were modified for clarity (adjustment of brightness and contrast) as needed.

Measurements

The measured parameters as well as measurement approaches were the same as in [25]; i.e., we focused on gall dimensions (length and width), infected cell dimensions, plasmodium diameter, and spore diameter. Additionally, we measured dimensions (length and width) of non-infected petioles. Galls and non-infected petioles were measured using the SZX12 stereomicroscope while the rest of the parameters were measured using the upright BX60 microscope (see above) and the QuickPHOTO MICRO 2.3 software. All measurements were done by a single person (V. K.) and compared with those published in previous studies.

DNA Extraction, Amplification, and Sequencing

Sterile tweezers and a scalpel were used to cut as much of the uninfected plant tissue off the gall as possible. The rest of the gall was surface sterilized in 10% SAVO (common household bleach, Unilever ČR Ltd., Czech Republic; 100% SAVO contains 47 g/kg, i.e., 4.7% sodium hypochlorite = NaClO) for 1 min and rinsed twice in sterile deionized water. The DNA was then extracted from the remaining tissue using DNeasy Plant Mini Kit (QIAGEN Inc., Venlo, The Netherlands) following the manufacturer’s instructions. To amplify the phytomyxid’s 18S rRNA gene sequence, a nested PCR with the following protocol was carried out. The reaction mixture (25 μl) consisted of 12.5 μl of Plain Combi PP Master Mix (Top-Bio s.r.o., Praha, Czech Republic), 1.5 μl of each primer (final concentration 0.6 μM), 0.8 μl of 20 mg/ml bovine serum albumin, 2 μl of the extracted DNA, and 6.7 μl of deionized H2O. For the first round PCR (95 °C for 5 min of initial denaturation, followed by 35 cycles of 95 °C for 35 s, annealing at 50 °C for 35 s, extension at 72 °C for 10 min, and 72 °C for 10 min of final elongation), universal eukaryotic primers MedlinA (CTGGTTGATCCTGCCAG) [39] and EK-1498R (CACCTACGGAAACCTTGTTA) [40] were used. For the second round PCR (95 °C for 5 min of initial denaturation, followed by 35 cycles of 95 °C for 35 s, annealing at 54 °C for 35 s, extension at 72 °C for 10 min, and 72 °C for 10 min of final elongation), set of primers specific for phytomyxids, Plas1f (TCAGTGAATCTGCGGATGGC) [16] and PHMX-1570R (GCKARTTGCAAGMSGCAAGC; redesigned from the primer Plas1r [16] to cover all Phytomyxea species), were used. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN Inc.). The purified products were then Sanger sequenced by Macrogen Europe Laboratory (Macrogen Inc., The Netherlands), using the 577F (GCCAGCAGCCGCGGT), 577R (ACCGCGGCTGCTGGC), 1055F (GGTGGTGCATGGCCG), and 1055R (CGGCCATGCACCACC) sequencing primers [41]. The newly determined sequence of the 18S rRNA gene of the phytomyxid symbiont of Halophila stipulacea has been deposited in GenBank under the accession number MN128644.

Molecular Phylogenetic Analysis

A dataset of aligned 18S rRNA gene sequences was created. It contained the newly determined sequence, 16 Phytomyxea sequences retrieved from GenBank, 12 environmental 18S rRNA gene sequences identified by BLAST to be affiliated with Phytomyxea, and 11 sequences of vampyrellids, Placopus, “novel clade 9,” and Penardia and its relatives, used as out-groups. The sequences were aligned using MAFFT [42] on the MAFFT 7 server (https://mafft.cbrc.jp/alignment/server/) with the G-INS-i algorithm at default settings, followed by manual correction and exclusion of poorly aligned sites in BioEdit 7.0.4.1 [43]. The final dataset contained 1725 aligned sites and is deposited as Supplementary file 1. Phylogenetic trees were constructed by maximum likelihood (ML) and Bayesian inference (BI) methods. The ML analysis was performed using RAxML 8.0.0 [44] under the GTRGAMMAI model, with 10 random taxon additions to generate starting trees. Bootstrap support was inferred from 1000 pseudoreplicate datasets. BI was performed using MrBayes 3.2.2 [45] under the GTR + I + Γ + covarion model. Two parallel runs of four MCMCs were run for 3,000,000 generations at which point the mean standard deviation of split frequencies based on the last 75% of generations was < 0.01. The trees were sampled every 500th generation. The first 25% of trees were removed as burn-in.

Results

Herbarium Material Examinations

We found the permanent slides from Marziano et al. [24] to be closely comparable with the galls collected by Vohník et al. [25] in Turkey as well as to the material collected in this study, in terms of both the endophytic infection pattern (Fig. 2a) and the spore size (Fig. 2b, c). However, it was impossible to reliably determine the frequency of the different spore arrangements due to the high spore density in the Marziano et al. [24] slides. On the contrary, the different spore arrangements were clearly recognizable in the micrographs of the recent findings by G. M. Gargiulo (Fig. 2d, e): most of the spores formed dyads or were single, which is in agreement with all published observations of this symbiosis in the Mediterranean (see above).

Fig. 2
figure 2

Micrographs of earlier findings of the phytomyxid infection in Halophila stipulacea in Sicily. a Transverse section of a gall on H. stipulacea examined by Marziano et al. [20]. b, c Micrographs of mature resting spores from the galls examined by Marziano et al. [20]. d, e Micrographs of mature resting spores from galls on H. stipulacea collected by G. M. Gargiulo in around 2015 near Tono (Casabianca), Sicily (Table 1). Note that most of the spores are arranged in dyads. Scale bars: a = 300 μm; b = 40 μm; c = 20 μm; d, e = unavailable

Sampling

Out of the four sampling campaigns, only two were successful in terms of phytomyxid infection findings (Table 1). In December 2015, no H. stipulacea populations were found at the localities visited. In September 2017, a population of H. stipulacea was discovered at one locality in Messina at the depth between 11 and 14 m with a relatively high phytomyxid colonization rate (Table 2). In May/June 2018, several new localities with H. stipulacea were found but there was no sign of the infection at any of them. At the locality in Messina where the infected plants had been found the previous year, many rhizomes were observed with the leaves broken off, but no galls or other signs of the infection were observed. However, in September 2018, the infection was detected at the same site again, although this time, the percentage of the colonization was slightly lower (Table 2). Water salinity did not differ between the two samplings in Messina in 2018 and was 40‰. The measured average water temperature at the collection site fluctuated between 17 and 23 °C, regardless of the season. The values represent isolated measurements and are greatly affected by the constantly changing sea currents, characteristic for the Strait of Messina ([46], Fig. 13).

Table 2 Phytomyxid infection levels in the seagrass Halophila stipulacea collected in this study in Messina (Sicily, southern Italy). Each year, three samples of H. stipulacea biomass were collected at the site with the presence of the phytomyxid infection (see Table 1) for an estimation of its intensity. For further details, see “Materials and Methods

When present at the site, the infection was immediately visible to the naked eye (Fig. 3a) and even its different developmental stages could be easily recognized by their different coloration (Fig. 3b), as previously described in “Materials and Methods.”

Fig. 3
figure 3

Morphological and anatomical changes in Halophila stipulacea petioles due to the phytomyxid infection. a Plants with visible phytomyxid infection (arrows) observed in situ. b A rhizome with different developmental stages of the infection, distinguishable by the coloration of the modified petioles/galls. c Morphology of a healthy H. stipulacea petiole (arrowhead) and a petiole with a very indistinctive infection (arrow), where the morphological changes are hardly noticeable. d Host cells infected with the phytomyxid lie in close proximity, forming characteristic clusters (arrows) located along the central vein of the petiole. e Exceptionally heavily infected petiole with three clusters (asterisks) of infected cells. f Sporogenic plasmodium and its cleavage into smaller plasmodia (asterisks), stained with safranin/Fast Green FCF. g A dark colored structure of unknown origin (asterisks) located close to the membrane (arrow) surrounding the newly forming resting spores, stained with safranin/Fast Green FCF. h A dark colored compact structure as above (arrow) in a cell filled with mature resting spores. i Dyads and single resting spores. j An illustrative example of a resting spore triad. k An illustrative example of a resting spore tetrad. Scale bars: b = 4 mm; ce = 2 mm; f = 200 μm; g = 20 μm; h = 50 μm; i–k = 10 μm

Stereomicroscopy and Light and Scanning Electron Microscopy

When the petiole was infected with the phytomyxid, its usually long and thin proportions (Fig. 3c) changed. According to the extent of the infection (i.e., the number of the infected host cells), the morphological modification of the petiole altered from hardly detectable (Fig. 3c) to the formation of the characteristic galls (Fig. 3b); i.e., the petiole was considerably shorter and swollen. The galls comprised the petiole tissue with one or typically two clusters of infected parenchyma cells, each located on one side along the central vascular tissues of the petiole (Fig. 3d). In rare occasions of exceptionally heavy infection, a third cluster was formed (Fig. 3e).

In agreement with Vohník et al. [25], all three morphologically distinguishable developmental stages of the infection were often found on the same rhizome with the youngest leaves showing signs of the earliest infection stage (white-colored galls containing sporogenic plasmodia) to the mature infection stage (dark colored galls filled with mature resting spores) usually being developed somewhere around the third youngest node (Fig. 3b). However, there were also cases (although very seldom) where the youngest leaves of an already infested rhizome seemed not infected.

Using light microscopy, in thin sections of the white-colored galls, each infected host cell could be easily localized by the presence of a sporogenic plasmodium causing a significant enlargement of the cell (Fig. 3f). The cytoplasm of the plasmodium often concentrated on the periphery of the host cell creating a hollow structure, and, in many cells, its cleavage into smaller polygonal to elliptical subunits was apparent (Fig. 3f) (also compare with [25]). In more mature galls, formation of resting spores could be seen. In each host cell, the spores were surrounded by a membrane and dark colored structures of an unknown origin were often observed close to the membrane (Fig. 3g). In the host cells filled with mature resting spores, these structures seemed more compact and were usually of a rounded shape (Fig. 3h). The observed resting spores formed mostly dyads (60%) or stayed single (31%) although some triads (6%) and tetrads (3%) were also present (Fig. 3i–k) (Table 3).

Table 3 Comparison of reports on phytomyxid infections in the seagrass genus Halophila (continuing from Vohník et al. [25])

During the determination of the phytomyxid colonization percentage in September 2018, few older detached leaves were noticed in the samples. Their gall tissue had already started to disintegrate, but the original infection pattern was still easily recognizable (Fig. 4a–d). Upon further examination, we discovered that the resting spores fell out from the decaying petioles not individually, but as massive aggregations surrounded by a membrane (Fig. 4d–g), leaving empty cavities where the infected enlarged cells had been once located (Fig. 4d, h).

Fig. 4
figure 4

Gall disintegration and spore release. ac Old H. stipulacea leaves with thick swollen petioles indicating former phytomyxid infection/galls. Asterisks mark empty cavities created by the release of spore aggregations into the environment. d Different stages of the gall emptying: spore aggregations (arrows) are visibly falling out of the damaged petioles, leaving empty cavities (asterisks) in the host tissue. e A SEM micrograph of mature phytomyxid spore aggregations (asterisks) still situated in the petiole tissue. f Individual spore aggregations after their release from a disrupted gall. g Each spore aggregation is visibly coated by a membrane (arrow). h A SEM micrograph of an empty gall with hollow cavities (asterisks) and visible imprints of formerly present enlarged infected host cells (arrow). Scale bars: a, b, f, h = 1 mm; c, d = 2 mm; e = 100 μm; g = 50 μm

Measurements

According to our measurements, well-developed galls cause shortening of the petiole by approximately two-thirds of its average length (i.e., 8.7 mm; n = 34, min. 6.0 mm, max. 11.7 mm) and almost doubling of its average width (i.e., 1.6 mm; min. 0.9 mm, max. 2.3 mm). The gall average dimensions measured in this study (3.2 × 2.6 mm) are very similar to those published in [25], although the resting spore diameter (5.5 μm) and dimensions of the infected cells (260.3 × 198.8 μm) were slightly larger here (Table 3). These numbers differ significantly from the dimensions characteristic for Tetramyxa parasitica as originally described in [30] (resting spore diameter 3.5 μm, plasmodium diameter 15–30 μm), further indicating that this phytomyxid is not T. parasitica (also see [35]).

Molecular Phylogenetic Analyses

The phylogenetic tree of Phytomyxea inferred from the 18S rRNA gene sequences is shown in Fig. 5. Phytomyxea was recovered as monophyletic with maximum support and split into three robust clades (bootstrap support, BS 100, Bayesian posterior probability, BPP 1), i.e., Plasmodiophorida, Phagomyxida, and the environmental clade “TAGIRI-5” (see [47]); the latter two appeared closely related, though the relationship was not highly supported (BS 71, BPP < 0.9). The newly determined sequence of the phytomyxid colonizing Halophila stipulacea clearly belonged to the clade “TAGIRI-5”, representing its first characterized member.

Fig. 5
figure 5

Phylogenetic tree of Phytomyxea, based on the 18S rRNA gene sequences, constructed by the maximum likelihood in RAxML (GTRGAMMAI model). Shadowed boxes highlight the three major clades of Phytomyxea. Values at branches represent statistical support in bootstrap values (RAxML)/posterior probabilities (MrBayes); support values below 50/0.90 are not shown or are represented by an asterisk. The newly determined sequence is indicated in bold

Discussion

The rediscovery of the phytomyxid symbiosis after 20+ years in the Strait of Messina suggests that either the respective phytomyxid is well established at least in some of the local H. stipulacea populations or it has been repeatedly introduced into this area, either as infected seagrass fragments or in the form of resting spores/spore aggregations. Indeed, the Strait of Messina is a relatively narrow but important seaway connecting the Eastern Mediterranean Basin with the Tyrrhenian Sea, so the possible repeated introductions could have happened through commercial and leisure traffic, similarly to the situation with H. stipulacea in the Caribbean [48], perhaps being facilitated by the notorious Strait currents periodically changing their directions and intensity [46] (http://www.correntidellostretto.it/). However, the observed absence of the characteristic galls at most localities with H. stipulacea investigated in this study suggests that the phytomyxid is either rare (as has been suggested for Plasmodiophora bicaudata [19]) or overlooked (similar to the case of Plasmodiophora cf. halophilae at the Turkish locality in the Aegean Sea [25]).

The fact that the symbiosis was very common in Messina (> 40% of the leaf petioles colonized) in September 2017, then totally absent in May/June 2018 and then again common (~ 30%) in September 2018 importantly adds to our so far very limited knowledge on ecology of this phytomyxid species as it indicates that its life cycle either is adapted to a particular life cycle stage of its plant host or is somehow influenced by the ambient water temperature. The host seems to be free of infection for most of the year as the phytomyxid remains dormant, probably in the form of environmentally resistant resting spores/spore aggregations. This apparent seasonality may explain the very low number of reports on its occurrence and the observed differences in colonization rates. In the same area in Sicily, Marziano et al. [24] reported less than 1% of the petioles colonized (with no details on the sampling date) whereas in this study, we found more than 40% of the petioles colonized in September 2017. Thus, our findings emphasize the need for periodic screening of the investigated localities and could serve as a warning against premature conclusions on the distribution and population dynamics of marine phytomyxid species.

According to the phylogenetic analyses based on the 18S rRNA gene sequence, the organism studied here is closely related to the phagomyxids, being a member of the clade “TAGIRI-5”, which comprises also environmental clone sequences from the East China Sea [49] and the coast of Japan [50]. The “TAGIRI-5” clade certainly represents at least a new genus of Phytomyxea and the organism studied by us represents its first characterized member. For its accurate taxonomic treatment, a morphological and phylogenetic comparison with Plasmodiophora halophilae is necessary. However, we were unable to obtain the type specimen of P. halophilae on loan from the Botanical Museum in Copenhagen, Denmark (i.e., the place where it was originally deposited by H. Jensen, see [29, 51]), and, unfortunately, it seems to be irretrievably lost. Moreover, to our best knowledge, no phytomyxid infection has been reported on Halophila ovalis in Java, Indonesia, since the paper by Ferdinandsen and Winge [29], so at present, such a comparison is impossible. The H. stipulacea phytomyxid should be also compared with Tetramyxa parasitica, despite T. parasitica seems to be specific only for salt-tolerant aquatic plants such as Ruppia and Zannichellia [32,33,34]) and certain Potamogeton species [52, 53]. Indeed, except the paper by Marziano et al. [24], to our best knowledge, it has never been reported on Halophila or any Mediterranean seagrass. These taxonomic issues will be eventually solved by molecular approaches, but at the moment, T. parasitica sequences are not available in the NCBI-GenBank database (accessed June 2019). This obviously holds true also for P. halophilae. Until then, for practical purposes, we suggest continuing to label the phytomyxid species associated with H. stipulaceaPlasmodiophora cf. halophilae” as initiated by Vohník et al. [25].

Based on the anatomy and morphology of the galls as well as the appearance and the arrangement of the spores, it is almost certain that the phytomyxid reported in [24, 25], and here represents the same species, thus extending its known distribution range from the northeastern part of the Eastern Mediterranean Basin to the borderline with the Western Basin. Since H. stipulacea is a well-known Lessepsian migrant and phytomyxids are obligate symbionts/parasites, there are two possible scenarios available to explain the origin of this phytomyxid symbiosis in the Mediterranean Sea: (1) the symbiosis arrived to the Mediterranean as a whole, i.e., both the host and its intracellular symbiont travelled through the Suez Canal together, or (2) the seagrass and the phytomyxid travelled separately, i.e., as phytomyxid-free seagrass fragments and environmentally resistant resting spores/spore aggregations, either in parallel or at various times, and formed the characteristic symbiosis only after the first contacts in the Mediterranean Sea. Alternatively, considering the predisposition of phytomyxids even for cross-kingdom shifts [16], Plasmodiophora cf. halophilae could have lived in the Mediterranean in symbioses with alternative hosts and switched to H. stipulacea once it arrived through the Suez Canal and colonized suitable habitats (although we consider this scenario unlikely). In any case, given the lack of the symbiosis at the site in Messina in May/June 2018 and its return in September 2018, it seems plausible that the phytomyxid has found favorable conditions for completing its life cycle even close to the current northwesternmost limit of the distribution of H. stipulacea in the Mediterranean [26, 27], from the release of the resting spores to the formation of the zoospores which colonize host tissues, eventually triggering the formation of the characteristic petiole galls full of the resting spores (see [10]). It would therefore be interesting to investigate the phytomyxid spore bank not only at the site in Messina but all along the coast of the Strait of Messina, perhaps using approaches similar to those common in terrestrial phytomyxid research [54, 55].

In May/June 2018, no galls were observed at the locality in Messina but many H. stipulacea rhizomes were observed without older leaves. Additionally, many already dead leaves mostly with galls at various stages of disintegration and releasing resting spores/spore aggregations were found at the same place in September 2018. It is unclear whether the observed leaf shedding was facilitated or even triggered by the phytomyxid infection at the point when the resting spores were mature and ready to disperse into the surroundings. Nevertheless, spore dispersal aided by the dead leaves would certainly be of a significant advantage in terms of both dispersal range and spore protection. The latter may be further enhanced by formation of the spore aggregations enveloped in a protective membrane as observed in this study (Fig. 4e–g). Some phytomyxids may, interestingly, cause increased uprooting of the host rhizomes [19, 56, 57], which can also facilitate the dispersal of resting spores. Such a phenomenon has however not been observed in the case of the H. stipulaceaPlasmodiophora cf. halophilae symbiosis.

Conclusions

Our study shows that the phytomyxid symbiosis on H. stipulacea reported in the Mediterranean Sea firstly in 1995 and secondly in 2017 may be actually more common than previously thought and that targeted research throughout the whole year is necessary to fully understand its distribution as well as the infection process of the respective phytomyxid. DNA-based investigations (including P. halophilae and T. parasitica) are needed to elucidate the identity of the phytomyxid and its taxonomic position among the already described marine species. Investigations into the phytomyxid spore bank, including areas in the northwest Mediterranean Sea where H. stipulacea is currently absent, may further expand our understanding of its population dynamics, dispersal modes, and future infection potential.