Description of Brooksia lacromae sp. nov. (Tunicata, Thaliacea) from the Adriatic Sea

Brooksia lacromae sp. nov. is described from zooplankton material collected at a marine monitoring station in the South Adriatic in the autumn of 2014. The description of both solitary and aggregate forms is given along with 18S rRNA and mitochondrial cox1 sequence data that provides strong evidence that both forms belong to the same species. The described species is morphologically markedly different from B. rostrata (Traustedt, 1893) and B. berneri van Soest, 1975, previously the only two species in the genus Brooksia. Genetic analysis based on 18S rRNA gene confi rmed distinctness of B. lacromae sp. nov. from B. rostrata (1.5% uncorrected pairwise distance). The appendicularian Fritillaria helenae Bückmann, 1924, so far known from the Atlantic only, was found in the same samples as B. lacromae sp. nov. Co-occurrence of B. lacromae sp. nov. with an Atlantic appendicularian suggests an Atlantic or Western Mediterranean origin for this new taxon.


Introduction
Thaliacea is a well investigated group, primarily because of their bloom potential and substantial grazing impact on phytoplankton communities (Madin & Deibel 1998;Andersen 1998).The last new species within the order Salpida were described 40 years ago (van Soest 1975), when, among other species, Brooksia berneri van Soest, 1975 was described.Brooksia berneri and Brooksia rostrata (Traustedt, 1893) are two morphologically very similar species that make up the genus Brooksia (van Soest 1975;Godeaux 1998).The blastozooid of Brooksia berneri is unknown or identical to that of Brooksia rostrata (van Soest 1975).Both species are known from tropical and temperate parts of the Atlantic, Indian and Pacific Oceans and their ranges seem to overlap (van Soest 1975).In the Mediterranean there have been only three records of Brooksia rostrata (Sigl 1912;Fedele 1926;Batistić et al. 2014)

R e s e a r c h a r t i c l e
urn:lsid:zoobank.org:pub:38CC4797-768A-44C5-8B85-EA3DCB6678CB berneri has not been recorded.The last record of Brooksia rostrata was of a single oozooid in 2008 at Lokrum station (Batistić et al. 2014;Fig. 1).After the discovery of Brooksia lacromae sp.nov. in 2014, the material from 2008 is re-examined in this paper and it proved in fact to be an individual of Brooksia lacromae sp.nov.
Brooksia lacromae sp.nov.was found in samples collected at the Lokrum station (South Adriatic, Fig. 1) in October 2014.The Lokrum station is located off the south-east Adriatic coast of Croatia where it is under the direct influence of incoming currents from the Ionian Sea.Such entering currents are known to modify Adriatic zooplankton community composition and to bring alien species of Atlantic/ Western Mediterranean or Eastern Mediterranean/Lessepsian origin into the Adriatic on a pluriannual scale (Batistić et al. 2014;Gačić et al. 2010;Civitarese et al. 2010).
The life cycle of salps consists of two alternating generations: the solitary asexual generation (oozooids) and the aggregate sexual generation (blastozooids), which morphologically differ greatly.Here, we describe both forms of Brooksia lacromae sp.nov.Given the considerable morphological differences between these reproductive forms, and the limited differences between Brooksia species in aggregate morphology, we also use genetic data to corroborate our descriptions.DNA sequences from both the nuclear ribosomal small subunit RNA (18S) and mitochondrial cytochrome oxidase subunit I (cox1) genes were generated, as these have previously been successfully used for integrative taxonomic research on closely related salp species (Goodall-Copestake 2014).Finally, we also discuss the potential origin of Brooksia lacromae sp.nov.

Collection of zooplankton samples
Zooplankton samples were collected on 3 Oct. 2014 at the Lokrum station (42°37'21" N, 18°06'05"E; European Journal of Taxonomy 196: 1-13 (2016) instrument (Seabird) from the surface to 90 m depth at every metre.The planktonic tunicate community composition was determined using an Olympus SZX-16 stereo microscope by analysis of entire zooplankton samples to the species level.The specimens of B. lacromae sp.nov.for morphological analysis were additionally stained with a few drops of 0.1% Janus Green dye, which made muscle bands clearly visible.Specimens were photographed using a DP25 (Olympus) digital camera mounted onto the stereo microscope and measured from the photographs using Cell^D software (Olympus).In addition to formalin-fixed samples, material for genetic analysis was taken with a 53 μm mesh net from 90 m depth to the surface.The plankton material was then filtered through a 53 μm mesh to remove sea water and subsequently preserved in 95% ethanol, for no more than 2 days.The ethanol sample was sorted for Brooksia lacromae sp.nov.oozooids and blastozooids and then cleaned of attached debris using dissection needles.Cleaned salps were placed in separate tubes containing pure acetone for long-term storage at -20°C.

DNA isolation, PCR and sequencing
DNA was isolated from one oozooid individual (branchial bar tissue) and one blastozooid individual (muscle and test tissue).About 1-2 mm 2 of the tissue was cut and used for DNA isolation.After removal of acetone and drying the tissue for 20 min at 55°C, tissue fragments were lysed for 2 hours at 55°C in 30 μl of a solution containing 10 mM Tris-HCl pH 8.0, 0.5% SDS, and 0.5 mM EDTA, with the addition of 1.5 μl of 20 mg ml -1 proteinase K (Fermentas).After lysis, the DNA was purified using the modified method described by Karatani et al. (2006) based on the anti-chaotropic effect of saturated (NH 4 ) 2 SO 4 solution on DNA molecules.The lysate was mixed with five times the volume of saturated ammonium sulphate water solution and put in hand-made spin columns.The hand-made spin columns with glass filters were made by making two holes with a red hot needle in a 0.5 ml polypropylene tube, one in the cap and the other in the bottom of the tube.Two glass filters, 4 mm in diameter each, were then put in every column.The glass filters were cut out of GF/F (Whatmann) filter using a flame-sterilised hollow punch tool.After binding of DNA to the glass filters, the columns were washed with 300 μl of a buffer made by mixing 10 ml of 10 mM Tris (pH 7.5) with 40 ml of 96% ethanol.After drying the columns by centrifugation at maximum for 3 min, the DNA was eluted with 35 μl of TE buffer pH 8.0 (10 mM Tris, 1 mM EDTA).The 18S rRNA gene was PCR amplified and sequenced in four overlapping fragments using the primer pairs app2f-uni800r, 500f-app1200r, oiko800f-1500r, and 1300f-n1800r (Table 1); the overlap between any two fragments was 200-400 base pairs.The cox1 fragment was amplified using primers coi-70f and coi-930r (Table 1), which were designed by aligning complete cox1 sequences of multiple tunicate species available from GenBank.The PCR was performed in a 100 μl PCR mix containing: 1 × PCR buffer, 0.2 mM of each dNTP, 3 mM MgCl 2 , 0.2 μM of each primer, 1.2 U of recombinant Taq polymerase (Thermo Scientific) and 2 μl of template DNA.All amplifications of 18S DNA were performed using a PCR programme with a 2 min denaturation step at 94°C, with 40 subsequent cycles of 94°C for 20 s, 50°C for 30 s, 72°C for 1 min, and a final extension step at 72°C for 5 min.The cox1 fragment was amplified using a PCR programme with a 2 min denaturation step at 94°C, with 40 subsequent cycles of 94°C for 20 s, 45°C for 2 min, 72°C for 3 min, and a final extension step at 72°C for 5 min.
PCR products were purified for sequencing using the same method as for DNA isolation and eluted in 17 μl of 10 mM Tris buffer (pH 8.0).
All PCR products were sequenced by the Macrogen company (South Korea) in both directions using the same primers as used for original amplification.Obtained 18S rRNA and cox1 gene sequences from an oozooid and a blastozooid were deposited in GenBank under accession numbers: KR057222 (oozooid 18S), KR057223 (blastozooid 18S), KT818685 (oozooid cox1) and KT818686 (blastozooid cox1).

DNA data analysis
Sequence assemblies from 18S overlapping fragments were made using the BioEdit software (Ibis Biosciences).Uncorrected pairwise distances were calculated between B. lacromae sp.nov.18S sequences and a B. rostrata 18S sequence obtained from GenBank, as well as between different B. lacromae sp.nov.sequences.Cox1 sequences were translated using ascidian mitochondrial genetic code to check for nuclear pseudogenes and no stop codons were detected.

Planktonic tunicate community composition
A total of 29 species of planktonic tunicates was recorded on 3 Oct. 2014 at the Lokrum station (Table 2).Out of 29 species, there were 23 species of Appendicularia, four of Doliolida and two of Salpida.The most abundant appendicularians were Oikopleura fusiformis Fol, 1872, Oikopleura longicauda (Vogt, 1854), Oikopleura parva Lohmann, 1896 and Fritillaria haplostoma Fol, 1872.The most abundant doliolid was Doliolina muelleri Krohn, 1852.The most abundant salp was Brooksia lacromae sp.nov.(0.2-0.9 ind.m -3 ), while Thalia orientalis Tokioka, 1937 was found in low numbers (0-0.1 ind.m -3 ).European Journal of Taxonomy 196: 1-13 (2016) ventral longitudinal muscle at their anterior part, while muscles VI and VII fuse with them posteriorly.Muscles II, III, IV and V converge ventrally towards the junction between muscle I and the branches of the ventral longitudinal muscle.Muscle II can be slightly fused with muscle I just before its connection with branches of the ventral longitudinal muscle.Endostyle straight.Stolon emerges from the test ventrally, slightly posteriorly from the nucleus.There is one test processus just posteriorly from the stolon (Fig. 4D).Muscles I-VII with 52-58 muscle fibers in total, measured laterally in two examined individuals.

Dextral individual
Mirror image of sinistral individual.

Etymology
Brooksia lacromae sp.nov. is named after the island of Lokrum near which it has been found.The Latin name of the island of Lokrum is Lacroma.

Holotype
One oozooid (14.8 mm body length, 23.9 mm total length including proboscis), collected from a 0-50 m depth layer on 3 Oct. 2014 in a 53 μm mesh plankton net.It is deposited in the Tunicata collection of the Croatian Natural History Museum under inventory number CNHM Inv.br.44/1.
The temperature average in the 0-50 m layer was 19.0°C, with a range between 16.2°C and 22.2°C, while the salinity average was 38.36, with a range between 37.79 and 38.68.In the 50-90 m layer the temperature average was 15.8°C, with a range between 15.5°C and 16.1°C, while the salinity average was 38.71, with a range between 38.67 and 38.77.

Remarks
Oozooid.Brooksia rostrata and Brooksia berneri oozooids are very similar, differing only in the fact that in B. berneri oozooid muscle I, joined with the intermediate muscle (im), is separated from muscle II and discontinuous mid-dorsally.Due to this fact we will only compare the B. lacromae sp.nov.oozooid to the B. rostrata oozooid.The Brooksia lacromae sp.nov.oozooid has one ventral longitudinal muscle which extends into the proboscis, while B. rostrata has two (Fig. 2).The proboscides in both collected oozooids broke off during collection, but were found in the sample (Fig. 2c).The proboscis of Brooksia lacromae sp.nov.seems to be thinner at the base than in Brooksia rostrata and in both species it contains two lateral longitudinal muscles.The ventral longitudinal muscle in B. lacromae sp.nov.has two small slit-like openings anteriorly, one below the endostyle and the other just anteriorly to it, partly overlapping with the anterior end of the endostyle.In Brooksia lacromae sp.nov., in contrast to B. rostrata, body muscles are not arranged in a barrel-like structure where muscles are perpendicular to the body axis dorsally, as well as ventrally, but they converge to the posterior third of the body.Only muscles I, VI and VII fuse with the branches of the ventral longitudinal muscle, while muscles II, III, IV and V converge to the connection between muscle I and branches of the ventral longitudinal muscle, without fusing with either.Only muscle II can sometimes be slightly connected with muscle I ventrally.The stolon in Brooksia lacromae sp.nov.seems to emerge from the test, slightly posteriorly from the nucleus (Fig. 4D), while in B. rostrata it emerges from the anterior part of the nucleus (Thompson 1948).
Brooksia rostrata the left intermediate muscle is discontinuous.Its dorsal and ventral counterparts both enter the anterior attachment processus.Because of this, the anterior attachment processus in B. lacromae sp.nov.possesses two muscles (IM1-a and IM2), while in B. rostrata it possesses three muscles (dorsal and ventral left intermediate muscle and right intermediate muscle).According to Thompson (1948), the right intermediate muscle of the sinistral individual of B. rostrata ends blindly, while after Tokioka (1954) and Godeaux (1998) it connects to muscle I.In B. lacromae sp.nov. the right intermediate muscle (IM2) ends blindly.

Genetic analysis
There were no differences between blastozooid and oozooid 18S sequences.Between two cox1 sequences there were 7 substitutions out of 837 nucleotides, which results in 0.84% uncorrected pairwise distance.Cox1 sequences were translated using ascidian mitochondrial code and there were no differences in amino acid sequence between them.The uncorrected pairwise distance between the Brooksia rostrata 18S sequence (HQ015403) and the Brooksia lacromae sp.nov.18S sequence (KR057223) was 1.5% (including gaps).Out of 26 differences in 1740 nucleotides between these two sequences, there were 21 transitions, 4 transversions and one single nucleotide gap.

Discussion
In the Mediterranean there are only two finds of Brooksia rostrata according to our knowledge.The first record was in 1911 on the eastern side of the Middle Adriatic (Sigl 1912) and the second from the Gulf of Naples (Fedele 1926).The find of B. rostrata in the South Adriatic in 2008 (Batistić et al. 2014) proved to be in fact an individual of B. lacromae sp.nov., after careful examination of one oozooid in poor condition.It thus seems that Brooksia species are rarely recorded in the Mediterranean.
Recent investigations have shown that entering currents, of either Atlantic/Western Mediterranean or Eastern Mediterranean origin, modify the composition of the gelatinous zooplankon community in the South Adriatic and each type of current brings different newcomers (Batistić et al. 2014).According to a recently postulated BiOS theory, the entrance of different water masses into the Adriatic depends on the direction of circulation of the North Ionian Gyre (Civitarese et al. 2010;Gačić et al. 2010).The finding of the appendicularian Fritillaria helenae Bückmann, 1924, so far only known from the Atlantic, in the same samples as Brooksia lacromae sp.nov., suggests that B. lacromae sp.nov. is of either Atlantic or Western Mediterranean origin.In addition to F. helenae, Kowalevskia oceanica Lohmann, 1899 and Fritillaria formica digitata Lohmann in Lohmann & Buckmann, 1926 were found in the same samples, which is their first peer-review published record from the Adriatic (Garić & Batistić 2013).Fritillaria fraudax Lohmann, 1896, a rare appendicularian in the Adriatic as well as in the Mediterranean (Skaramuca 1980;Fenaux 1967;Fenaux et al. 1998), was found in high numbers, which might also be related to the increased inflow of water of Atlantic or Western Mediterranean origin.Curiously, the oozooids of Brooksia lacromae sp.nov.were only found in a 53 μm mesh net, which suggests that the filtration speed in a 200 μm mesh net may be too high to preserve the delicate oozooid body.
The morphological differences between Brooksia rostrata and Brooksia lacromae sp.nov.are quite high for congeneric salp species (Godeaux 1998).The number of oozooid body muscles is the same in both species but their dorsal fusing pattern and overall arrangement is markedly different.In B. rostrata body muscles are parallel to each other and they fuse ventrally with the two longitudinal ventral muscles, forming a barrel-like pattern.In B. lacromae sp.nov.body muscles converge ventrally to the posterior third of the body and, except for muscles I, VI and VII, they don't fuse with the single longitudinal ventral muscle or its branches.The blastozooids are much more similar.There are only two differences between B. rostrata and B. lacromae sp.nov.blastozooids.In B. rostrata the muscle IM1 enters the anterior attachment processus and the muscle IIR-b enters the posterior attachment processus, while in European Journal of Taxonomy 196: 1-13 (2016) B. lacromae sp.nov.only a branch of the first intermediate muscle (IM1-a) enters the anterior attachment processus (ap1) and the muscle IIR-b does not enter the posterior attachment processus (ap3).Tokioka (1954) described a long testicular processus emerging from the posterior end of mature blastozooids of B. rostrata.No such structure could be seen in examined B. lacromae sp.nov.blastozooids, but that is perhaps due to their immaturity.
The 18S rRNA gene is very conservative and tends to underestimate the number of species when used in biodiversity studies (Tanga et al. 2012;Raupach et al. 2010).It is generally used for phylogenies of distant species or higher taxonomic groups.The pairwise distance of 1.5% between B. rostrata and B. lacromae sp.nov.18S sequences was higher than most intrageneric species distances of Thaliacea (Govindarajan et al. 2011), supporting the designation of B. rostrata and B. lacromae sp.nov.as separate species.In addition to 18S sequence data, cox1 sequences from B. lacromae sp.nov.are also provided.Although only one oozooid and one blastozooid sequence were generated, the pairwise difference between them was consistent with expectations for intra-specific divergences in salps (cf.Goodall-Copestake 2014).There are only a few published thaliacean cox1 sequences (Yokobori et al. 2005;Leray et al. 2013;Goodall-Copestake 2014), mainly because of their high evolutionary rate, which makes common universal cox1 primers unusable.The cox1 sequences generated for the present study will aid in the future identification of B. lacromae sp.nov.as well as help with further refinements of Thaliacea-specific cox1 PCR primers.
In the last couple of years two new species of gelatinous zooplankton were described from the Adriatic Sea (Garić & Batistić 2011;Piraino et al. 2014), while one species was re-established more than 100 years after its first description (Batistić & Garić 2016).This incidence of new species of gelatinous zooplankton in the Adriatic is likely partly due to climate change, with more and more alien species entering the Adriatic (Batistić et al. 2014), and partly as a consequence of poor quality investigations of marine zooplankton at the species level (Batistić & Garić 2016).In order to improve our ability to detect climate-induced changes in marine ecosystems in the future, we suggest more research effor t be placed on taxonomically challenging groups such as the gelatinous zooplankton described here.
(aggregate form; Figs 3, 4A-C) Sinistral individual -Four muscles on the left side, three muscles on the right side.Dorsal IM1 continues ventrally as the ventral muscle IM1, without extending to the anterior attachment processus (ap1).A branch of dorsal muscle IM1 (IM1-a) extends to the anterior attachment processus (ap1).Dorsal muscles IR, IIR and IIIR branch dichotomously.Dorsal muscle IR-a continues ventrally as ventral muscle IR.Dorsal muscle IR-b continues ventrally as muscle IIR-c.A small branch from muscle IR-b/IIR-c enters the lateral attachment processus (ap2).Dorsal muscle IIR-a continues ventrally as ventral muscle IIR-a.Muscle IIR-b ends blindly on the ventral side of the animal without entering the posterior attachment processus (ap3).Dorsal and ventral muscles IIIR enter the posterior attachment processus (ap3) without joining.Muscles IVL-a and IIIR-a extend from muscles IVL and IIIR, respectively, towards the nucleus.Endostyle curved anteriorly.Muscles IL-IIIL, IR-a,b and IIR-a,b,c with 3 muscle fibers each in all examined individuals.Muscle IVL with from 3 to 9 muscle fibers, muscle IIIR with from 5 to 7 muscle fibers.

Fig. 4 .
Fig. 4. Brooksia lacromae sp.nov. A. Photograph of the ventral side of the dextral blastozooid.B. Photograph of the dorsal side of the sinistral blastozooid.C. Photograph of the lateral side of a blastozooid.D. Detail of the ventral posterior end of an oozooid.All indivuduals were dyed with Janus Green dye.Abbreviations: st = stolon; tp = test processus.
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Table 1 .
PCR primers used for amplification of B. lacromae 18S rRNA and cox1 gene fragments.