First records of hydroid epibionts on the introduced macroalga Gracilariaparvispora in the Mexican Pacific

Abstract Background The red macroalga Gracilariaparvispora is an introduced species in the Mexican Pacific. To date, there are no published studies on its sessile epibionts, including the hydrozoans and bryozoans, which are the dominant epibionts on macrophytes and of significant biological and economic interest. New information This study provides insight into the faunal diversity of hydroids growing on G.parvispora. A total of 185 thalli from both herbarium specimens and field samples collected from five sites in La Paz Bay were revised. Each thallus size and the presence of hydroid epibionts in each thallus region were registered. Eight different hydrozoan taxa were growing on the red macroalgae, including the first recorded observation of Obeliaoxydentata in the Gulf of California. The sizes of the collected thalli were mostly under 7.0 cm, the maximum number of taxa per thallus was three and the thallus region containing the highest number of epibionts was in the middle. Significant differences were observed amongst the lengths of thalli with and without epibionts. The thalli with epibionts were larger than the thalli without epibionts. Similarly, significant differences were observed amongst the months. The pair-wise test revealed that each month exhibited distinctive epibiont taxa when compared to the others. This study highlights the lack of information on these associations. Further research is needed to understand whether introduced macroalgae can bring non-native epibiont species to an area.


Introduction
Epibiosis is defined as an association between two or more living organisms, whereby one associate, the basibiont, provides a suitable surface for the settlement of the other(s), the epibionts (Wahl 1989, Wahl 2010).In marine ecosystems, macroalgae as basibionts provide a complex substrate by creating microhabitats where epibionts can attach, grow and reproduce (Schmidt and Scheibling 2006).Colonial invertebrates, commonly sessile epibionts of macroalgae, mostly belong to the phyla Cnidaria and Bryozoa, which present a planktonic larval phase that can adhere to a basibiont and form temporary or permanent colonies (Ryland 1962, Nishihira 1967, Stricker 1989, Connell 2000, Ryland 2005, Hiebert et al. 2020).The encrusting or erect colonies remain physically and physiologically connected through identical modular units, termed zooids in bryozoans and polyps in cnidarians (Mackie 1997, Hiebert et al. 2020).The epibiont cnidarians highlight the benthic polyps of class Hydrozoa, which are referred to as hydroids and these may have life cycles with a medusa phase, predominantly planktonic (Collins 2002, Oliveira et al. 2006, Cartwright and Nawrocki 2010), while the bryozoans have only benthic zooids (Bock 1982).
Several studies have observed hydrozoans and bryozoans growing on macroalgae (cf.Manríquez and Cancino 1996, Oliveira and Marques 2011, Cunha et al. 2017b, Carral-Murrieta et al. 2023) and have also highlighted the preference of some of these invertebrates for specific species and macroalgal morphologies (Gallardo et al. 2021, Carral-Murrieta et al. 2024).However, the ecological role and impact of introduced macroalgae as basibionts in coastal marine ecosystems are poorly understood.It has been found that introduced macroalgae-dominated systems have shown varying effects on local biodiversity and that epibiosis can be a mechanism to facilitate the invasion of epibionts as well (Arnold et al. 2015, Lazzeri andAuker 2022).Therefore, it is important to identify and inventorise the epibionts species growing on introduced macroalgae to monitor and manage the impact on local biodiversity.Additionally, macroalgae and colonial invertebrates are of biological, scientific and social interest due to their positive or negative economic impact on the pharmaceutical, food, biotechnology, fishing and aquaculture industries (cf. Grohmann (2008), Muñoz-Ochoa et al. (2010), Wood et al. (2012), Mouritsen (2013), Pereira (2018), Pinteus et al. (2018), Kintner and Brierley (2018), Ciavatta et al. (2020), Banagouro et al. (2022)).
Gracilaria parvispora, also known as limu ogo or long ogo, was described from Kaneohe Bay, Oahu, Hawaiian Islands, USA (Abbott 1985, Lembi andWaaland 1988) and, since then, it has been recorded in Korea, Japan andChina (Kim et al. 2008, Guiry andGuiry 2024).However, the origin of G. parvispora in Hawaii is unclear (Abbott 1999, Nelson et al. 2009) and its epibionts are also unknown.It is one of the three most sought-after seaweeds for food in the Hawaiian Islands and a potential source of agar (Abbott 1999, Krueger-Hadfield et al. 2016).
In this context, the present study aimed to analyse the hydrozoans associated with an introduced macroalgae, G. parvispora and determine potential assemblages of these epibionts according to the macroalga's morphological characteristics, based on an analysis of herbarium and collected thalli in a subtropical bay of the Gulf of California, Baja California Sur (BCS).

Herbarium specimens
Gracilaria parvispora dried specimens were obtained from the Phycological Herbarium of the Autonomous University of Baja California Sur (FBCS) and were collected in La Paz Bay: El Mogote, La Concha and El Caimancito, which are beaches frequented by tourists (Table 1, Fig. 1, Suppl.material 1).

Field samples
Based on previous reports of G. parvispora in La Paz Bay, BCS, five sites were visited, including disturbed and undisturbed environments.The port of San Juan de la Costa, the Roca Fosfórica Mexicana phosphorite mine at San Juan de la Costa (ROFOMEX SJC), La Concha, the port of the Autonomous University of Baja California Sur Pichilingue (UABCS Pichilingue) and Punta Diablo (Table 1, Figure 1, Suppl.material 1).In Punta Diablo, natural substrates were present without direct contact with nautical traffic.The other four sites had various artificial substrates and were exposed to nautical traffic or anthropogenic activities.ROFOMEX SJC and the port of San Juan de la Costa are adjacent to a phosphorite mine with a daily production of 6,000 tonnes (Servicio Geológico Mexicano 2018).Its port is located 2.8 km north of the fiscal dock in La Paz Bay.La Concha is a tourist beach with hotel activities and a recreational diving company.UABCS Pichilingue is located 3.2 km north of the Pichilingue's port, where tourist and commercial ships arrive (Fig. 1).Following periodic changes in surface temperature and the entrance and retirement of tropical waters in La Paz Bay and Gulf of California (Santamaría-del-Ángel et al. 1994, Flores-Ramírez et al. 1996, Guevara-Guillén et al. 2015), the sites were visited in spring (transition period between cold and warm waters), winter (cold waters) and summer (warm waters) during the annual cycle 2021-2022.The complete macroalgal thalli were randomly sampled manually by the same team and using a knife or scraping artificial or natural substrates (n ≥ 10) by snorkelling and scuba diving in sites with depths of more than three metres.The samples were fixed in 96% ethanol for morphological analysis.
First records of hydroid epibionts on the introduced macroalga Gracilaria ... with a sounding weight and calibrated line marked from 0 to 5 m in 20 cm increments, while at the dive sites, it was measured with a dive computer.Thalli were identified according to morphology (Fig. 2) following Abbott (1985), Dreckmann (1999) and García-Rodríguez et al. (2013) descriptions.In the laboratory, the length (cm) and presence of cystocarps of herbaria and collected thalli of G. parvispora were registered.
Each thallus was divided into three equal regions (basal, middle and apical) and the presence or absence of hydrozoans on the thallus was recorded.The basal region consisted of the first third closest to the disc and part of the stem, the middle region included the central part of the alga and the last third of the thallus from the middle part to the tips of the alga was catalogued as the apical region (Fig. 2).
Epibionts were identified with the support of taxonomic descriptions and compilations available in literature (e.g.Millard (1975)  For each taxon, we provided the material section (locality, depth, data, habitat, data generalisations), diagnosis and notes [figure, type locality, references for a detailed description of the species, taxonomic status with a unique and persistent identifier that assures the taxonomic quality control denominated "AphiaID" (Vandepitte et al. 2015) and remarks (only for taxa with additional information)].In the material examined, we included the sampling site and date, temperature (°C), salinity (PSU), depth (m) and presence of epibionts in the specific macroalgal regions.Descriptions, taxonomic status and dichotomous key are included only for specimens identified to species level.
The significant differences in size between thalli with and without epibionts were tested by one-way permutational multivariate ANOVA (PERMANOVA) using untransformed data and Euclidean distances.Additionally, PERMANOVA was used to determine whether epibiont assemblages differed significantly amongst: 1) month, 2) cystocarp and noncystocarp thalli and 3) basal, middle and apical thallus regions.The presence-absence data matrix was analysed using the Jaccard similarity measure with 9999 permutations and significance was set at p < 0.05.When a significant effect was found, post hoc paired comparisons between factor levels were performed (Anderson 2001, McArdle andAnderson 2001).Percentage similarity analysis (SIMPER) was also used to determine the contribution of species to within-group similarity and between-group dissimilarity (Clarke et al. 2014).Statistical analysis was performed in PRIMER v.6 using the PERMANOVA+ add-on software (Clarke and Gorley 2006, Anderson et al. 2008).

Gracilaria parvispora and hydrozoan epibionts
Thalli of G. parvispora were mainly found in sandy substrates and can be on pebbles, rocky and bivalve shells (Fig. 3a).The thallus is cylindrical, except in the branches, which are flattened to cylindrical, usually with three orders of branches, from 0.5 to 4.0 mm in diameter and present irregular dichotomous branches, sympodial without a defined main axis.Thallus has a flaccid consistency and its colouration can vary: yellow, green, red or brown.There are large medullary cells from 90 to 280 mm in diameter, with two cortical cells surrounding the medullary cells.Cystocarps are present from 2.0 to 5.0 mm in diameter (Fig. 3b), as well as chain carpospores of ovoid to the slightly spherical shape from 15 to 35 μm in diameter (Fig. 3b).
The total number of thalli analysed was 185, of which 10 were from herbarium specimens.The length ranged from 1.4 to 36 cm, with 88% of the samples under 7.0 cm.Eight percent presented cystocarps (15 thalli) and 22% presented hydrozoan epibionts (41 thalli), which were found in thalli sizes from 1.6 to 17.0 cm and a maximum of three taxa per thallus were recorded.Sixty-three percent of the thalli with epibionts had two species of epibiont hydroids (Fig. 4).
Six species and two genera of the Hydrozoa epibionts were identified and were observed only in the collected thalli.In addition, no bryozoans were found in the collected thalli, but First records of hydroid epibionts on the introduced macroalga Gracilaria ... some belonging to the Gymnolaemata class, order Cheilostomatida, were observed on four herbarium thalli (Fig. 5).It should be noted that the bryozoans observed were morphologically damaged and it was not possible to identify them at a lower level of order.As this was the first record of epibionts in this macroalga, the epibiont hydroids were integrated into a dichotomous key and taxonomically described.Percentage and size (cm) of Gracilaria parvispora thalli with the number of epibiont taxa found.First records of hydroid epibionts on the introduced macroalga Gracilaria ...
Detailed description in Calder (1983), Calder (1997) Internodes with annulations at the base and one distal apophysis alternately given off the hydrothecal pedicel.Hydrotheca short and conical, with diaphragm oblique and margin entire.Hydrothecal pedicel with annulations along its whole length.With conical gonothecae with a short distal neck, arising from the base of the hydrothecal pedicel or the axis of the main stem and branches.
Remarks.It is now widely accepted and supported that the traditional concept of O. cf.dichotoma (cf.Cornelius (1995)) comprises multiple cryptic lineages (Calder 2013, Calder et al. 2014, Cunha et al. 2017a, Calder et al. 2019, Cunha et al. 2020) and in the eastern Pacific, affinities still need to be determined between local populations ( Calder et al. 2019), mainly because their lineages are not distinguished from each other by morphometric analyses (Cunha et al. 2020).Therefore, molecular studies will be necessary to delimit the eastern Pacific lineages.Taxonomic status.Accepted.AphiaID 766210.

Remarks.
Colony morphology and size support evidence that this species differs from Obelia bidentata Clark, 1875.Previous studies have discussed the reason for the recognition of the species, supporting the correct identification of the species ( Calder 2013, Calder 2019, Calder et al. 2019).This species is 1-60 mm tall, with predominant sizes from 1 to 10 mm (Calder et al. 2019, Calder 2020, Calder et   First records of hydroid epibionts on the introduced macroalga Gracilaria ... Notes: Fig. 6g Type locality.Ecuador: Santa Elena Bay (Calder et al. 2009).

Remarks.
Recent morphological studies support the validity of species with sufficient support.Therefore, we follow the morphological evidence indicated in Mendoza-Becerril et al. (2020).

Identification keys
Dichotomous key of Hydrozoa epibionts of Gracilaria parvispora Hydrothecal margin with about 11 to 16 cusps, deeply cut teeth separated by U-shaped incisions; margin scalloped in cross-section, with V-shaped pleats extending inwards towards hydrothecal cavity; each pleat originating at the apex of each tooth and continuing downwards to the upper part of hydrothecal wall.

-
Hydrothecal margin with about 15 -20 long cusps, slightly rounded, with deep, rounded spaces between them, which alternately show a slight difference in depth so that an indistinct paired arrangement occurs.Hydrothecal margin with about 15 -20 long cusps, slightly rounded, with deep, rounded spaces between them, which alternately show a slight difference in depth so that an indistinct paired arrangement occurs.
SIMPER (similarity percentage) analysis results demonstrated that taxa accounted for the most similarity within each month and the most dissimilarity between months.

Discussion
A total of eight hydrozoan epibionts species were recorded for the first time in G. parvispora thalli.One of the most notable differences in the presence of epibionts was the quantity found in field and herbarium thalli.The latter yielded fewer epibionts, possibly due to the preparation of the thalli before being fixed, as these were rinsed free of any sand or debris, without emphasis on the conservation of the epibiont fauna.Therefore, epibionts with erect growth and calcareous or chitinous exoskeletons are only sometimes preserved when dried since they become brittle and are often lost in herbarium samples (M.A.M.-B.and K.L.-C.pers.obs.); for example, calcareous bryozoans were observed in the herbarium thalli.In the collected thalli, no other encrusting epibionts were observed, such as bryozoans or sponges, even though literature shows evidence that these epibionts co-exist (cf.Maggioni et al. ( 2020)) and are considered dominant epibionts (Altuna 1994, Gappa andSabattini 2007) due to their ability to survive the spatial competition.This is a primordial characteristic in colonial organisms since they adapt under selective pressure to environmental changes (Jackson 1977, Gili et al. 2000).
The hydrozoans were mainly found in the middle region of the G. parvispora thallus, in contrast to the benthic Sargassum species and Cystoseira amentacea (C.Agardh) Bory, where the basal region hosted the highest number of hydrozoan species (Fraschetti et al. 2006, Carral-Murrieta et al. 2024).This contrast may be related to the preference for sandy substrates of G. parvispora as opposed to benthic Sargassum species and C. amentacea for rocky reefs, as well as the competition for the surrounding fauna and the growth of other algae.Moreover, colonial invertebrate larvae avoid settling in regions of the alga with members of competitively dominant species; therefore, the epibionts tend to settle on the youngest parts (Stebbing 1971, Grosberg 1981).For this reason, the epibiont fauna is usually smaller in the apical area because of the high growth rate and renewal of filaments in this zone in the algae of the genus Gracilaria (Molina-Vargas and Álvarez-León 2014).Furthermore, the richness and abundance of hydroids also depend on the macroalgal morphology.For instance, highly branched macroalgae have many micro-habitats that facilitate hydroid settlement and persistence in macroalgae such as Cystoseira barbata (Stackhouse) C.Agardh, C. amentacea and Sargassum spp.(Faucci andBoero 2000, Carral-Murrieta et al. 2024), while G. parvispora has a single dominant axis, usually with three branching orders.

Conclusions
This study demonstrated that the macroalga G. parvispora is a basibiont hosting colonial sessile epibionts, with the most frequent group being hydrozoans.This is the first time that the associated fauna of this macroalga has been studied and it provides essential information on the taxonomy and diversity of their epibionts.However, since macrophytes are potential vectors for species introductions in other regions (Kuhlenkamp and Kind 2013), further studies on non-native or invasive macroalgae and their epibionts are needed to assess whether these algae are conducive for the introduction of bryozoan or hydrozoan species into the local fauna.It is also important to assess whether the presence of colonial epibionts is directly related to their geographical distribution, whether they exhibit opportunistic settlement on the substrate (Oliveira and Marques 2011) or whether they prefer specific lineages of macrophytes for their development ( Munari et al. 2015).Having established the basis for locating the macroalga in La Paz Bay and identifying its epibionts, it is possible to propose a standardised methodology for analysing whether the diversity and abundance of the epifauna depend on the macroalgae's morphology, as Gan et al. ( 2019) suggested for the epifauna associated with different macroalgae.The methodology of future studies should also consider the incorporation of the variation of environmental parameters.
, Mendoza-Becerril et al. (2020)) and the nomenclature used was based on a study by Maronna et al. (2016) for Leptothecata hydroids and the World Register of Marine Species (WoRMS Editorial Board 2024) for other hydroids.After the analysis, the specimens (algae and invertebrates) were deposited in the Macroalgae Laboratory from Centro de Investigaciones Biológicas del Noroeste, S.C.

Figure 2 .
Figure 2. Gracilaria parvispora.a Field sample of La Concha, scale equals 2.0 cm; b Herbarium specimen under code FBCS2490, El Caimancito, scale equals 2.0 cm; c Scheme with regions for recording epibionts.