The marine sponge Hymeniacidon perlevis is a globally-distributed invasive species

In Elkhorn Slough, a tidal estuary draining into Monterey Bay, California, the intertidal is occupied by a conspicuous orange sponge known by the name Hymeniacidon sinapium. This same species is found in the rocky intertidal zone of the outer coast of California, and is described herein from subtidal kelp forests of Southern California. Farther afield, morphologically and ecologically indistinguishable sponges are common in estuaries and intertidal areas in Asia, Europe, South America, and Africa. Here I use morphological, ecological, and genetic data to show that these sponges are all members of the same globally-distributed species, which should be known by the senior synonym H. perlevis. Though previous authors have remarked upon the morphological, ecological, and/or genetic similarity of various distant populations, the true scope of this sponge’s distribution appears to be unrecognized or unacknowledged in the literature. Limited larval dispersal, historically documented range expansion, and low genetic variation all support a hypothesis that this sponge has achieved its extraordinary range via human-mediated dispersal, making it the most widely-distributed invasive sponge known to date. Declarations Conflicts of interest/Competing interests: none to declare Availability of data and material: All raw data is included as supplementary files; georeferenced collection data is available as a supplementary .xls file; genetic data are archived at Genbank; specimen vouchers are archived at the California Academy of Sciences and at the Natural History Museum of Los Angeles; specimen photos will be made available as supplementary files, are also archived by the associated museums in GBIF, and are posted as georeferenced data on iNaturalist.org. Code availability: n/a


Introduction
forth other names for these distant populations. For example, de Laubenfels described a 93 sponge he named Hymeniacidon sinapium from California in 1930 (de Laubenfels 1930Laubenfels , 94 1932. He acknowledged that "it is doubtful whether this is a new form", and went so far 95 as to suggest that species with the names "sanguinea, luxurians, caruncula, heliophila, 96 sinapium, and perhaps even more species" are in fact synonyms. Consistent  When describing H. sinapium in California, de Laubenfels remarked on its impressive 104 ecological breadth. He described it as abundant in the "surf-beaten" intertidal throughout 105 Southern California, but also the most abundant sponge on the oyster beds in Newport 106 Bay (de Laubenfels 1932). He reported only one sample from subtidal depths, but his 107 subtidal sampling was limited, primarily via trawling. In contrast to this abundance in 108 Southern California, de Laubenfels was only able to locate a single specimen of this 109 species in Central California. This is notable because he was based at Hopkins Marine 110 Station in Monterey Bay (Central California) in the 1920s, and this was the area that he 111 studied most comprehensively at that time. A monographic report on Elkhorn Slough, 112 which drains into Monterey Bay, was published in 1935: it reports 4 species of sponges in 113 the estuary, but none similar to H. sinapium (MacGinitie 1935). This makes it unlikely 114 that this species was present in large numbers in Central California in the 1920s. 115 Subsequently, however, it has become a common species in intertidal portions of Elkhorn 116 Slough, which drains into Monterey Bay (Wasson et al. 2001), and it is also known from 117 Tomales Bay in Northern California (Wasson et al. 2001; Fuller and Hughey 2013). 118 Morphological (Sim 1985) and genetic (Hoshino et al. 2008) comparisons later confirmed 119 that a common Hymeniacidon in Korea, Japan, and China were the same species as those 120 in California, so it was proposed that H. sinapium was introduced to California from Asia 121 with oyster mariculture (Fuller and Hughey 2013). Though this is certainly possible, the 122 data I compile here illustrates that it may also be non-native in Asia. This species has 123 been said to occur in the Mexican Pacific (Hofknecht 1978) and the Galapagos Islands 124 (Desqueyroux-Faúndez and Van Soest 1997) as well, but genetic data are not yet 125 available from those populations. 126 127 The final species to consider, H. heliophila (Wilson 1911), is ascribed a substantial range 128 in the Western Atlantic, from the Gulf of Maine to Brazil ( (Wilson 1911), it is also said to be 131 very common in the Caribbean (Diaz et al. 1993). A recent paper also found that an 132 indistinguishable sponge was the most common intertidal sponge present in the Bahía 133 San Antonio, Argentina, (Gastaldi et al. 2018 placed in a 1.5 ml microcentrifuge tube with household bleach for several hours, until 177 tissue appeared to be dissolved. With the spicules settled at the bottom of the tube, the 178 bleach was then pipetted off and replaced with distilled water; this was repeated several 179 times (I found that 2-3 water changes were sufficient for visualizing spicules with a light 180 microscope, but removing all the salt from the sample for other downstream applications 181 required 5 or more rinses and worked best when the final ones were done with absolute 182 ethanol). In some cases, samples were centrifuged at low speed to reduce settling time 183 between rinses, though this increased the proportion of broken spicules.

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Spicules were imaged using a compound triocular scope and pictures were taken using a 186 D3500 SLR camera (Nikon) with a NDPL-1 microscope adaptor (Amscope). Pictures of 187 a calibration slide were used to determine the number of pixels per mm, and 20-30 188 spicules were then measured using ImageJ (Schneider et al. 2012). Spicules length was 189 determined in a straight line from tip to tip, even when spicules were curved or bent.

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Spicules were selected randomly, so as to get an unbiased estimate of size distributions.

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All raw data are available as supplementary indicates which were included in each analysis, and explains the reasons why any were 247 excluded. Some reads were included in the phylogenetic analysis, which could tolerate 248 unequal read lengths, but not the haplotype network, which included only samples with 249 complete data over the entire alignment. Sequence alignments were produced in Codon 250 Code v.9 (CodonCode Corporation). Haplotype networks were produced using the  it was largely buried in sediment, with projections extending into the water column. In 281 contrast to its prevalence on the Southern California mainland, I did not find it at any 282 island sites. This difference seems unlikely to be due to dispersal limitation because 283 island and mainland sites have high connectivity (Watson et al. 2010). It is more likely 284 due to the ecological differences between sites: none of the island sites investigated had 285 areas with the fine silty sediment where the sponge was most common on the mainland. 286 Though silty sites at the islands may simply have been unsampled in this survey, it is 287 likely they are less common than on the mainland. For example, satellite data show that 288 particles at the islands are less prone to resuspension by wave action (Freitas et al. 2017 Gross Morphology of kelp forest samples 307 All but one of the newly collected samples were found embedded in sediment with 308 irregular projections extending into the water column. These projections varied from 309 stout cone-shaped or bag-shaped oscula to long, tendril-like digitations. One sponge was 310 found unburied, growing on rock. It lacked projections and instead resembled 311 Halichondria panicea (its identity was confirmed with spicule and DNA data, presented 312 below). All samples had a fleshy consistency, with the rock-dwelling sponge somewhat 313 firmer. Color varied from yellow to yellowish-orange in the field. Field photos are 314 available for 8 samples in the supplementary data accompanying this paper, and are also 315 available at iNaturalist.org. 316 317 I was interested in whether these sponges could be identified in the field and therefore 318 monitored using roving diver surveys or photo transects. These samples were collected as 319 part of an ongoing project to characterize the diversity of kelp forest sponges, with over 320 500 samples collected to date. This is one of the first surveys of sponges in California via 321 SCUBA, and the first with extensive field photos of specimens that have also been 322 analyzed morphologically. Though the bulk of these data will be published elsewhere, 323 comparisons to date indicate that H. sinapium is the only sponge in these habitats that 324 grows by extending irregularly shaped projections out of silty sediment. Though this 325 morphology is certainly known from other species, H. sinapium was the only sponge with 326 this morphology found within the sampling effort shown in supplementary table 1. This 327 indicates that this morphology, when found in the Southern California kelp forest, is 328 strongly suggestive of the presence of this species. The most similar species found to date 329 is Polymastia pachymastia: as the name suggests, this sponge is covered in nipple-like 330 projections. This sponge was also found covered in sediment, with only the projections 331 visible. However, these projections tend to be uniform in shape and regularly spaced in P. 332 pachymastia, which contrasts with the irregularly spaced and morphologically various 333 projections seen in H. sinapium. The projections are also nearly white in P. pachymastia, 334 while they vary from yellow to nearly orange in H. sinapium. The rock-dwelling H. 335 sinapium found at Carpenteria Reef, however, would be more challenging to identify 336 from field photos, as it is very similar to other Halichondridae found in the survey.

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Spicular morphology 339 I characterized the spicules of 9 samples to confirm their identity and compare them to 340 published data. All spicules were styles: tapered to a point at one end, and rounded at the 341 other. Width was usually uniform over the entire length, but a small minority had faint 342 swelling at or near the rounded end. This was manifest as a very weak swollen head 343 including the end (similar to the head of a match), or more commonly as a swollen band 344 near the head end (like a bead on a string). Most were somewhat curved or bent. The 345 skeleton of one sample was investigated further using hand-cut sections cleared with 346 Histoclear. Spicules in perpendicular sections through the choanosome formed wavy, 347 meandering tracts, the largest of which were about 30 μm wide. Spicules were also found 348 outside the tracts pointing in all directions (referred to as a "confused" arrangement in 349 sponge taxonomy). Surface sections revealed that the ectosome of the sponge was filled 350 with spicules that appeared to be tangential (parallel to the sponge surface) and also 351 "paratangential" (at an angle to the surface of less than 90 degrees). These spicules were 352 in messy bundles that formed an approximate mesh on the surface of the sponge. locus had more than 2 sequences available in Genbank from any of these taxa. 373 Preliminary phylogenies indicated that sequences of Hymeniacidon flavia were more 374 closely related to the clade containing my target species than anything else in Genbank.

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When available, these sequences were included for comparison. 376 377 Figure 2 shows the haplotype networks for the three loci with the most data. is less divergence than at the ITS and cox1 loci, indicating lower power to discriminate 445 among species on a per-base basis. The aligned region is 3 times longer than cox1 and 7 446 times longer than ITS, however.

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The D3-D5 region of 28S also allowed for an interesting comparison (figure 3 (which increased mortality), sponge larvae swam for a maximum of 24 hours and some 511 were still exploring the benthos when the experiment was terminated at 68 hours. These 512 data are consistent with the larval ecology of other sponges (Maldonado 2006). It 513 therefore seems unlikely that the larvae of this species have exceptionally higher 514 dispersal than to other sponges. 515 516 The data do not seem sufficient to form a strong hypothesis about the native range of this 517 species. It seems unlikely to be California, as almost no genetic variation was found at 518 the ITS or cox1 loci in that region. Moreover, the species has likely undergone range 519 expansion in California between the 1920s and the present. Europe is perhaps the most 520 likely source, as we know it was present there in the early 1800s. The genetic diversity at 521 cox1 in Portugal seems notably higher than the diversity at the ITS locus in Asia, though 522 better support would clearly come from comparing data from the same locus. 523 524 Future work will be needed to understand what impact this species has on host 525 ecosystems. Its abundance in some habitats seems to make impacts likely, if for no other 526 reason than the occupation of space (Wasson et al. 2001). It is also notable that it has 527 successfully colonized the kelp forests in California, which have been relatively resistant 528 to invasion (Steneck et al. 2002