On the Presence and Ubiquity of the Exotic Batophora (J. Agardh) in the Mar Menor Lagoon (SE Spain)

: We ﬁrst report the presence of a species of the genus Batophora in the Iberian Peninsula, in the Mar Menor lagoon (Murcia, SE Spain). We detected this macroalga in November 2021. However, according to some observations, it could have been present in the lagoon at least since 2016, being described as a “rare” form of Dasycladus vermicularis . A further survey made during May 2022 conﬁrmed the presence of this species in much of the perimeter of the Mar Menor. Morphologic analyses are not conclusive regarding the species description, but most traits point to Batophora occidentalis or Batophora occidentalis var. largoensis . A consensus sequence from the rbcL barcode gene was compared by the Blastn tool showing its closeness to other Batophora specimens, yet the speciﬁc level was unresolved. The recent conﬁrmation of a Batophora species in a littoral lagoon from Formentera (Balearic Islands) with similar morphologic characteristics could indicate that putative introductions are connected. Further morphologic and genetic analyses are required in order to know the origin and expansion of this genus along the Mediterranean coasts.


Introduction
The Mar Menor is a hypersaline coastal lagoon located in the southeast of the Iberian Peninsula (Figure 1), considered to be one of the largest coastal lagoons in the Mediterranean, with a surface of 135 km 2 and a maximum depth of 6.5 m [1]. At present, its salinity ranges from 39 to 46 psu, and its water temperature ranges from 11 • C in winter to 30 • C in summer [1,2]. Its seabed is mainly covered by soft substrates, with few rocky areas, combined with monospecific or mixed-macrophytes meadows of Caulerpa prolifera and Cymodocea nodosa [3,4].
This lagoon is a singular environment, with an elevated ecological and economical importance [5][6][7]. It has been protected as a Natural Park, a Ramsar international site, and a Site of Community Importance (SCI) integrated in the Natura 2000 Network, among others, and includes 18 habitats of European interest, according to the EU Habitats Directive [5]. It is the habitat of some emblematic and protected species, such as the macrophytes Ericaria amaentacea and Cymodocea nodosa; the fishes Hippocampus guttulatus, Syngnathus abaster, and Aphanius iberus; the sponges Tethya aurantium and Tethya citrina; and the mollusk Pholas dactylus [8]. Currently, the Mar Menor acts as a reservoir for the "Critically Endangered" fan mussel Pinna nobilis, which colonized the lagoon in the mid-1980s from the Mediterranean Sea [9]. However, it is also important as the habitat of some target species for fisheries, such as Penaeus kerathurus, Sparus aurata, or Engraulis encrasicolus, among others [6].
Despite its importance and protection, the Mar Menor has suffered several impacts related to anthropogenic activities, such as mining, agriculture, and urban development [5,7,10,11]. In fact, in recent years it has suffered some events of high eutrophication (in 2015-2016, 2019, and 2021), some of them being associated with extreme rainfalls, which increased the inflow of water with a high concentration of nutrients, organic matter, and sediments into Figure 1. Location of the Mar Menor lagoon and sites where Batophora has been observed. Green circles mark sites where Batophora was detected in the present study (size indicates abundance of this species, see Material and Methods); green crosses indicate presence, while red crosses mark studied sites where this species was not found. The black arrow marks the Rambla del Albujón watercourse outlet, whereas blue arrows show the three inlets connecting the lagoon and the Mediterranean Sea.
Despite its importance and protection, the Mar Menor has suffered several impacts related to anthropogenic activities, such as mining, agriculture, and urban development [5,7,10,11]. In fact, in recent years it has suffered some events of high eutrophication (in 2015-16, 2019, and 2021), some of them being associated with extreme rainfalls, which increased the inflow of water with a high concentration of nutrients, organic matter, and sediments into the lagoon [2,12]. These eutrophication episodes generated anoxia and massive mortality of marine organisms [9,12].
Initially, the lagoon was naturally connected to the Mediterranean Sea by three inlets, but in 1973 the widening and enlargement of the main channel increased the connectivity of both water masses, diminishing the Mar Menor salinity and facilitating the introduction and expansion of some Mediterranean species, such as Caulerpa prolifera, Pinna nobilis, Cotylorhiza tuberculata, and Rhizostoma pulmo [1,13]. Another cause of regression in this lagoon is the appearance and expansion of alien species. Thus, vessel traffic and aquaculture, among others, have acted as vectors for the introduction of species that are also cited as Figure 1. Location of the Mar Menor lagoon and sites where Batophora has been observed. Green circles mark sites where Batophora was detected in the present study (size indicates abundance of this species, see Material and Methods); green crosses indicate presence, while red crosses mark studied sites where this species was not found. The black arrow marks the Rambla del Albujón watercourse outlet, whereas blue arrows show the three inlets connecting the lagoon and the Mediterranean Sea.
Initially, the lagoon was naturally connected to the Mediterranean Sea by three inlets, but in 1973 the widening and enlargement of the main channel increased the connectivity of both water masses, diminishing the Mar Menor salinity and facilitating the introduction and expansion of some Mediterranean species, such as Caulerpa prolifera, Pinna nobilis, Cotylorhiza tuberculata, and Rhizostoma pulmo [1,13]. Another cause of regression in this lagoon is the appearance and expansion of alien species. Thus, vessel traffic and aquaculture, among others, have acted as vectors for the introduction of species that are also cited as exotic in the Mediterranean Sea, such as Mnemiopsis leidyi, Bugulina stolonifera, Branchiomma boholense, Cerithium scabridum, Fulvia fragilis, Bursatella leachii, Callinectes sapidus, Styela canopus, and Palisada maris-rubri [13][14][15][16][17][18].
Some of these exotic species could survive and adapt to this new habitat, colonizing it and negatively affecting its biodiversity, which makes it crucial to detect and monitor any new introduction in the lagoon. In the present work, we first report the detection of a new exotic green macroalga of the genus Batophora J. Agardh in the Mar Menor lagoon. We have morphologically characterized our specimens and sequenced the rbcL barcode gene. Through a rapid assessment method, we have also mapped and quantified its abundance along the perimeter of the lagoon.
Further studies which derive from this work will include a complete sampling of the lagoon for estimating the expansion and possible effects of this species in order to recommend control measures for this invasion, if necessary. Moreover, and due to its characteristics, we consider the Mar Menor lagoon as a good case of study for evaluating the presence, development, and consequences of exotic species, which could be incremented due to the effects of climate change.

Materials and Methods
We photographed and collected specimens of Batophora in Punta del Galán (37. In each of these localities, we sampled in shore strands which were 15 m wide and 200 m long (<1 m depth) ( Figure 1). Abundance was calculated according to the density of Batophora thalli by using four semiquantitative categories, as follows [19]: 1: Rare and sparse clumps (<15 cm in diameter) separated from each other by more than 5 m. 2: Frequent clumps and decimetric patches separated from each other by less than 5 m. 3: Decimetric patches and clumps separated by less than 1 m. 4: Patches only separated by a few centimeters, resulting in the appearance of a discontinuous belt.
Fresh specimens were immediately transported in plastic bags to the laboratory, where they were described and photographed using a microscope (Olympus BH2, Olympus Corporation, Tokyo, Japan) fitted with a camera (Leica DFC 480, Leica Camera AG, Wetzlar, Germany).
For DNA analysis, samples were washed in distilled autoclaved water in order to remove salts and sand. The epiphytes were removed using sterilized tweezers.
DNA was extracted according to CTAB protocol (0.1 M Tris HCl, 0.05 M EDTA, 1.5 M NaCl, 0.05 DTT and 2% CTAB) [20]. PCR amplifications of samples were carried out using the VWR Red Taq DNA polymerase Master Mix with a final volume of 50 µL per reaction. The rbcL (ribulose-1,5-biphosphate carboxylase/oxigenase large subunit) gene was amplified using the pair of primers rbcL B (5 -ATGTCACCACAAACAGAAACTAAAGCA-3 ) and rbcL Q (5 -GATCTCCTTCCATACTTCACAAGC-3 ) [21]. Amplification was conducted using a LifeExpress Classic BIOER thermal cycler (Life Express, Hangzhou Bioer Technology Co., Ltd., Zhejiang, China). PCR reactions were performed following Zechman [21], increasing to 35 cycles in order to obtain a greater DNA quantity. Then, amplification products were assessed on 1.5% TAE 1X agarose gels and were purified for sequencing with GeneJET Gel Extraction Kit (Thermo Scientific) according to the instructions. Finally, PCR products were Sanger sequenced by an external laboratory (Macrogen, Madrid, Spain).
The forward sequence and the reverse complement of the reverse sequence were aligned in order to generate a consensus sequence. The consensus sequence was deposited in the NCBI database with the Accession Number ON971347, and voucher specimens were deposited in the ABH (herbario oficial institucional de la UA). Gaps and sequence discordances were fixed by analyzing the electropherograms. The obtained consensus sequence was compared to the NCBI database by the Blastn tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (accessed on 20 June 2022). Finally, a phylogenetic tree was constructed through the Fast Minimum Evolution model [22], including representative specimens from the main genera of Dascycladales found in the NCBI database.

Results
A Batophora species was detected in fourteen localities throughout the perimeter of the lagoon (Figure 1) on multiple substrata such as pebbles, mollusks such as Hexaples Water 2022, 14, 2909 4 of 10 trunculus and Pinna nobilis, and artificial substrata (plates of PVC, ropes, etc.). Specimens form aggregates of 5-40 cm in diameter found in sandy shallow bottoms along with Cymodocea nodosa and Caulerpa prolifera meadows. Other species present in the same areas were Acetabularia acetabulum, Caulerpa cylindracea, Chaetomorpha linum, Chondria capillaris, Cladophora spp., Ulva spp., and Ruppia cirrhosa.
Batophora aggregates were more abundant in the east of the lagoon, with higher abundances in the northeastern localities forming shallow, discontinuous belts. These belts were distributed right on the edge of the lagoon along the shore, being replaced by Cymodocea and Caulerpa meadows seawards (Figures 1 and 2).

Results
A Batophora species was detected in fourteen localities throughout the perimeter o the lagoon (Figure 1) on multiple substrata such as pebbles, mollusks such as Hexapl trunculus and Pinna nobilis, and artificial substrata (plates of PVC, ropes, etc.). Specimen form aggregates of 5-40 cm in diameter found in sandy shallow bottoms along with Cy modocea nodosa and Caulerpa prolifera meadows. Other species present in the same area were Acetabularia acetabulum, Caulerpa cylindracea, Chaetomorpha linum, Chondria capillari Cladophora spp., Ulva spp., and Ruppia cirrhosa.
Batophora aggregates were more abundant in the east of the lagoon, with higher abun dances in the northeastern localities forming shallow, discontinuous belts. These bel were distributed right on the edge of the lagoon along the shore, being replaced by Cym docea and Caulerpa meadows seawards (Figures 1 and 2). In the eastern localities, some clumps of Batophora were floating, being carried awa by the waves and currents, and some of them were returning to the sea bottom or foun as debris accumulated on the beach (Figure 3).
Batophora was not detected in the southwest of the lagoon, where opportunistic alga (Ceramium deslongchampsii, Chaetomorpha linum, Chondria capillaris, and Cladophora spp thrive. In the eastern localities, some clumps of Batophora were floating, being carried away by the waves and currents, and some of them were returning to the sea bottom or found as debris accumulated on the beach (Figure 3).
The thallus of our specimens is greenish, 30-50 mm long with whorled axes, forming a cylinder of 3-5 mm in diameter arising from a holdfast with rhizoids ( Figure 4). Each whorl consists of six to nine ditrichotomous branchlets which fork 3-4 times, decreasing in diameter and becoming hyaline from the second or third fork ( Figure 5). The main axis where branchlets are inserted is 400-500 µm in diameter. The first internode of the branchlet is 650-900 µm long and 175-250 µm wide. Whorls were separated ca. 1.5 mm from each other in the middle part of the axis. Fertile axes with 3(-2) gametophores in the distal part of the basal internode inserted by means of a very short pedicel. Fertile gametophores are spherical with 400-650 µm in diameter, or oval, being 350-500 µm wide and 550-620 µm long. Gametophores are grouped in the upper (1/5-1/8) part of the main axis (Figures 4-6). Gametangia are spherical and are 59-68 µm in diameter.     The thallus of our specimens is greenish, 30-50 mm long with whorled axes, forming a cylinder of 3-5 mm in diameter arising from a holdfast with rhizoids ( Figure 4). Each whorl consists of six to nine ditrichotomous branchlets which fork 3-4 times, decreasing in diameter and becoming hyaline from the second or third fork ( Figure 5). The main axis where branchlets are inserted is 400-500 µ m in diameter. The first internode of the branchlet is 650-900 µ m long and 175-250 µ m wide. Whorls were separated ca. 1.5 mm from each other in the middle part of the axis.      After considering most genera of Dasycladales from the NCBI database, the molecular analysis includes our sequence in the genus Batophora (Figure 7). However, our sequence (consensus sequence of 1338 bp) is identical to those from B. oerstedi and B. occidentalis, preventing any distinction at the species level. After considering most genera of Dasycladales from the NCBI database, the molecular analysis includes our sequence in the genus Batophora (Figure 7). However, our sequence (consensus sequence of 1338 bp) is identical to those from B. oerstedi and B. occidentalis, preventing any distinction at the species level.

Discussion
The genus Batophora J. Agardh is reported in the western tropical and subtropical Atlantic [23][24][25], the Canary Islands [26], and the Mexican Pacific [27]. Three species or varieties of Batophora are recognized [28]

Discussion
The genus Batophora J. Agardh is reported in the western tropical and subtropical Atlantic [23][24][25], the Canary Islands [26], and the Mexican Pacific [27]. Three species or varieties of Batophora are recognized [28]: B. oerstedii J. Agardh, B. occidentalis (Harvey) S. Berger and Kaever ex M.J. Wynne, and Batophora occidentalis var. largoensis (J.S. Prince and S. Baker) S. Berger and Kaever ex M.J. Wynne. The size of the algae, the location of the whorls along the main axis, the position and size of the gametophores, as well as the size and shape of the gametangia have been the most widely used characters with a taxonomic value. However, there is no consensus on how these characters define the different species and varieties [28][29][30][31][32].
Our specimens show gametophores with a diameter compatible to that described for any species of Batophora [28,32]. On the other hand, the presence of axes with oval as well as spherical gametophores along with spherical gametangia would bring our specimens closer to B. occidentalis, whereas the insertion of the gametophores exclusively in the distal part of each first whorl branchlet internode points to B. occidentalis var. largoensis [28]. Molecular data include our specimens in the genus Batophora, but the results are not conclusive at the species and variety level, due to the lack of molecular data for B. occidentalis var. largoensis. Furthermore, rbcL barcode gene sequences are identical for both species (B. occidentalis and B. oerstedii). All in all, the lack of morphological data consensus regarding the ability to discriminate between species of Batophora, along with the few molecular data available, highlight the need for further research on these issues.
As for habitat preferences, according to Prince and Baker [30], B. occidentalis var. largoensis (as B. largoensis) is more typical of exposed environments, while B. occidentalis and B. oerstedii would be more related to mangrove or lagoon environments [32,33]. Despite this, mixed populations with the three taxa have been found in mangroves and other communities of protected coastal environments [28] where this genus can be very ubiquitous [34]. In our case, this alga is well-established in a large part of the Mar Menor, even forming discontinuous inner belts in the shallowest landside zones, where it is the dominant macrophyte in many cases. However, these "Batophora belts" are usually replaced by mixed meadows of Caulerpa prolifera and marine angiosperms (Cymodocea nodosa, Ruppia cirrhosa) in the "seaside" just a few meters beyond (pers. obs.). This suggests that this alga avoids canopy-forming communities well, as it has been observed in other exotic species [35][36][37]. This alga was not detected in the SW of the lagoon, probably because this area is affected by the Rambla of Albujón, the main watercourse draining into the lagoon, which discharges large amounts of nutrients and sediments, especially during extreme storm events, such as those which took place in 2019 and 2021 [2,38]. This discharge has affected the S-SW of the Mar Menor, creating extensive eutrophic and hypoxic zones in its vicinity [9]. This could be the reason for us to only find opportunistic ulvophycean and red "turf-like" algae in SW localities, as Batophora does not tolerate high levels of eutrophication [33], while it seems to show a high dispersal ability, by means of those "floating clumps".
A population of this genus has recently been discovered in a coastal lagoon connected to the sea in the island of Formentera (Balearic Islands), where it experienced a rapid bloom [39]. The characters analyzed in our work (habit, gametophore size, gametophore distribution, gametangia size) largely overlap with those reported in the population of Formentera except for the presence, in our case, of some fertile axes with distinctly oval mature gametophores and nondetection of gametophores in the second whorl branchlet internode ( Figure 5, Table 1). Table 1. Comparison between the main characters found in our specimens and those found in Ballesteros [39].

Main Observed Characters
This Study Ballesteros, 2020 Given the phenotypic plasticity of Batophora [28], and the overlapping in morphologic traits, it is likely that both populations could be linked to the same introduction event. This argument would be strengthened because both detections are practically simultaneous. In our case, this species may have been mistaken for "rare" forms of Dasycladus vermicularis (Scopoli) that Krasser detected since approximately 2016 during a severe anoxic event that affected mixed meadows of Caulerpa prolifera and Cymodocea nodosa (Giménez-Casalduero, F and Rubio, I.; pers. com.) [40].
Dasycladus C. Agardh is frequent both in the tropical and the warm temperate seas, including the Atlantic and the Mediterranean. Just like Batophora, it is also reported in shallow protected habitats such as lagoons and mangroves [41][42][43]. In spite of the high macroscopic resemblance between Batophora and Dasycladus, some morphological characteristics of Batophora (such as the larger distance between whorls and the presence of several gametophores in each whorl branchlet) properly separate this genus from Dasycladus. The easy confusion with Dasycladus vermicularis and the detection of vegetative thalli attributable to Batophora species almost 20 years ago could underestimate the presence of Batophora in the Mediterranean Sea [44].
In any case, the ecological similarity of the Mediterranean systems where Batophora is detected with those tropical or subtropical systems where this alga probably comes from, including its thermal range [28,45,46], makes it plausible for a future expansion of Batophora throughout the Mediterranean Sea, at least in warm and sheltered enclaves. Seasonal monitoring and mapping, together with genetic studies of Mediterranean, Canarian, and tropical populations, are essential to better understand the origin and potential expansion of this "new" introduction.

Conclusions
This study shows the first occurrence of Batophora species in the Iberian Peninsula (Mar Menor, SE Spain), which had probably been confused with Dasycladus vermicularis since 2016. Its ubiquity along much of the perimeter of the Mar Menor, together with the recent expansion of this alga in other Mediterranean lagoons, show its high expansion capability, acting as a potential invader. Further morphological and molecular data are necessary to better know the species identity and its possible origin.