Discussion

Botryllus schlosseri is a Mediterranean sea/European Atlantic shallow water (down to 200 m depth) marine invertebrate (Berrill, 1950; López-Legentil, Turon, & Planes, 2006; Paz et al., 2003; Reem et al., 2017) that has become a cosmopolitan alien species on man-made submerged hard bottom substrates, inhabiting primarily marinas and harbors all over the northern and southern hemispheres’ temperate zones (Berrill, 1950; Lambert, 2001, Ben-Shlomo et al., 2001, 2010, Reem et al. 2013a,b, 2017). This species is also known as a fouling pest species tagged with economic costs in aquaculture (Arens et al., 2011; Carver et al., 2006). Traits like fast adaptation to man-made environmental conditions (Lambert & Lambert, 2003; Lambert, 2001) and enhanced mutation rates that increase genetic variability in newly established populations (Reem et al., 2013a) further contribute to B. schlosseri’s invasiveness success in a way that upon the establishment of pioneering colonies, this species quickly spreads to become one of the common species in the hard bottom invertebrate consortia (Lambert & Lambert, 1998; Lambert & Lambert, 2003; Locke, Hanson, MacNair, & Smith, 2009; Martin, LeGresley, Thorpe, & McCurdy, 2011). Many of the Botryllus schlosseri populations beyond the Mediterranean/European Atlantic coasts are established for more than five decades (e.g. in Africa [Millar, 1955] and South America [Orensanz et al., 2002]) and up to a century and more, as in the US east coast (Gould, 1841), California (Van Name, 1945) and New Zealand (Van Name, 1945). Puget Sound populations are probably among the youngest regional populations of this invasive species. Despite the early mentioning of B. schlosseriin a naval shipyard in the Seattle area (US Navy, 1951), this species has not been recorded in civil marinas for several decades. Indeed, the partial absence in our 1999 survey may indicate a very recent establishment and coincides with Cohen et al. (1998), who found B. schlosseri in Des Moines and Shelton, but not in Edmonds. Thus, the Puget Sound provides a unique opportunity to study relatively recent (ca. three decades) established B. schlosseri populations.
The results of the present study reveal significantly differentiated populations on a spatial and temporal scale. Moderate genetic drift (ranging from 0.027 to 0.071) within the populations and limited gene flow (up to 0.031) between locations were also noticed. Population clustering showed that the Des Moines and Shilshole populations are assigned to different clusters, while the Edmonds and Shelton populations represent a mix of the clusters. The low number of microsatellite alleles found in the Puget Sound populations compared to other sites worldwide, was remarkable.
The absence of B. schlosseri in two surveys conducted in Edmonds marina in 1998 (Cohen et al., 1998) and 1999 (this study), put this marina as a candidate for the most recent established site (out of the four studied marinas) for the B. schlosseri Puget Sound populations. Four years later (2003 sampling session), the allelic indices of this presumed newly established population significantly differed from those of the subsequent sampling period (two years later). In contrast, the Shilshole populations showed fewer fluctuations in the allelic indices between the sampling points, despite only a single colony found in 1999. This indication of a stable population is further supported by genetic drift, which is much lower in Shilshole compared to the other three sites. The genetic indices showed diverse behaviors in the studied marinas. While the allelic richness (AR) and gene diversity (He) were highly fluctuating in Des Moines, Edmonds and Shelton, these two indices were slowly declining over time in the Shilshole marina. The two northernmost marinas, Edmonds and Shilshole, showed a peak in AR during the 2005 sampling period, while in Des Moines and Shelton, the highest numbers were observed in 2018 and 2003, respectively. Only in Shilshole, the peaks of He (year 2003) and private allelic richness (year 2007), did not coincide with the AR. The differences in the genetic indices propose that even close populations exhibit distinct genetic patterns. Yet, Shelton had by far the lowest values in genetic indices, all indicating its remoteness.
Heterozygote deficiency and significant inbreeding coefficients (Fis) found in all Puget Sound populations are in line with previous studies on the U.S. west coast (Karahan et al., 2016; Reem et al., 2013a; Stoner et al., 2002) and worldwide populations (Ben-Shlomo et al., 2010; Lacoursière-Roussel et al., 2012; Paz et al., 2003; Reem et al., 2017; Reem et al., 2013b). Temporal fluctuations in allelic richness seem to be characteristic of B. schlosseri populations (this study; Karahan et al., 2016; Reem et al., 2013a) and differ from long-term trends in isolated populations of other organisms, where a decreasing (García-Navas et al., 2015) or increasing (Jason Kennington et al., 2012) allelic richness was observed.
On the U.S. west coast scale, the four studied Puget Sound populations are clustered together, despite more than 120 km separating the two furthest populations. In contrast, the Moss Landing (Karahan et al., 2016) and Santa Cruz (Reem et al., 2013a) populations in California were always assigned in separate clusters, despite them being just 20 km apart. This attests that, despite significant Fst and Dest values, and different AR peak timings, the Puget Sound populations on the west coast level remain genetically close. It is further of interest to find that Moss Landing populations are genetically closer to the Puget Sound populations than to the Santa Cruz populations, as revealed by the Netstruct clusters and the gene flow charts, a result supported by Reem et al. (2013a) assumption that Santa Cruz populations are isolated. Additionally, null alleles were frequent in both, Puget Sound and Moss Landing (Karahan et al., 2016), but not in Santa Cruz populations (Reem et al., 2013a). Year 2007 was marked with the highest gene flow between the Puget Sound and Moss Landing, a year following a severe flooding occurring in Moss Landing, where B. schlosseri was temporarily eradicated from that marina (Karahan et al., 2016). Thus, genetic flow originating from the Puget Sound might have its footprint in the recolonization of the new population in Moss Landing marina.
On a global comparison, the Puget Sound’s B. schlosseripopulations present one of the lowest allelic richness and gene diversity. These low numbers, even when compared to remote populations such as New Zealand (Ben-Shlomo et al., 2001) and South America (Ben-Shlomo et al., 2010), further illustrate the late establishment of the Puget Sound populations, and can be explained by the limited time for the accumulation of mutations and inflow of genetic material from far away populations. Further, B. schlosseri microsatellite loci exhibit disparate repertoires of allele sizes and allele richness in different populations, worldwide. While BS-811 has numerous alleles in all locations, other loci, like PBC-1 reveal fewer alleles in regions invaded recently by B. schlosseri colonies. Also, the amplification success of B. schlosseri microsatellites shows local differences, even when following the same protocols. In the Puget Sound populations, the amplification success of BS-8, BS-9 and PB-41 was relatively low (up to 30 % missing data), whereas the amplification of those loci in samples from the Israeli coast resulted in no failures (S. Tamir, pers. Comm.). Moreover, the native European populations (Reem et al., 2017; Reem & Rinkevich, 2014) revealed more alleles per any specific microsatellite locus, further supporting the Mediterranean/eastern Atlantic origin notion for B. schlosseri .
Studying newly established B. schlosseri populations for a period of 19 years in the Puget Sound revealed highly fluctuating patterns in genetic indices such as allelic richness (AR), gene diversity (He), inbreeding coefficients (Fis) and population structures (Fst), showing a significant deviation from the Hardy-Weinberg equilibrium in all populations. Due to these fluctuations, no temporal trend could be observed, and it is suggested, that despite remarkable variations between the different sites, the Puget Sound populations remain isolated and still closely related. A comparison on a worldwide level revealed a considerably lower number of alleles in the Puget Sound populations, supporting the recent introduction hypothesis.