Results
Altogether, 533 tissue samples were collected (521 analyzed) within a
period of 19 years, between 1999 and 2018 in four locations at the Puget
Sound area, WA, USA. Detailed allele frequencies of all eight
microsatellite loci are shown in Suppl. Table A.2. A two-tailed t-test
between the Fst values (without and with ENA correction for null
alleles) was non-significant (p=0.463), indicating that null alleles do
not influence the results. The inbreeding coefficient (Fis) was highly
significant for every population.
Local scale
Des Moines marina
In total, 190 tissue samples from seven sampling dates (years 1999-2018;
Suppl. Table A.3) were analyzed. All eight microsatellite loci were
polymorphic, summing up to 71 alleles. About one third of total alleles
(23/71), appeared in only a single sampling period, eight private
alleles were found in year 2018, three in each of the sampling years
1999, 2003 and 2013, and two in each of the sampling years 2001, 2005
and 2007. Null alleles were detected in all loci, with high occurrence
(> 0.1) in PB-41 (0.284), BS-811 (0.255), PB-49 (0.229),
BS-8 (0.18), PB-29 (0.175) and BS-9 (0.126). The genetic diversity
indices are summarized in Table 1 and reveal fluctuating allelic
richness and significant heterozygote deficiency, suggesting that the
populations are not in Hardy-Weinberg equilibrium (HWE). The highest
peak in allelic richness was in 2018, followed by another peak in 2003,
both of which coinciding with the peaks in private allelic richness.
The Mantel test showed no significant correlation between Nei’s genetic
distance and the time between the different sampling periods (Suppl.
Table A.4). The Des Moines populations were overall significantly
(p=0.001) differentiated (Table 2). Interestingly, the populations from
1999-2007 exceeded the 2013-2018 population’s Dest with 0.064 (p=0.001)
and 0.024 (p=0.062), respectively. The estimated genetic drift (Fs’) was
further stronger between 1999-2007 (0.076) than between 2013 and 2018
(0.048). Pairwise population differentiation (Fst) values showed that
only the pairs 2005/2013, 2005/2018 and 2007/2013 were not significantly
different (Suppl. Table A.5A). This lack of similarities between the
years contradicts the BAPS analysis, which revealed only a single
cluster. Yet, two clusters were elucidated by STRUCTURE (Fig. 2A). In
1999 and 2001, cluster 1 (red in Fig. 2A) was dominant, whereas the
later years were more mixed, with some slight dominance of cluster 2
(yellow in Fig. 2A) in 2007 and 2013.
Shilshole marina
In total, 152 tissue samples were analyzed from Shilshole marina.
Collections were performed seven times during 1999 -2018, but year 1999
was not included in the analysis as only a single colony was observed
(not collected). It is also noted that year 2005 sampling yielded with
just 13 samples, despite increased collection efforts. All
microsatellites were polymorphic, representing however, only a single
BS-8 allele in five of the sampling periods (2001, 2003, 2005, 2007 and
2018), and just one allele of BS-9 in 2005. A total of 63 microsatellite
alleles were found, with 15 private alleles, occurring just in a single
sampling period (Suppl. Table A.3). Four private alleles occurred in
2018, followed by three in years 2013 and 2007, two in years 2005 and
2003 and a single private allele in 2001. Null alleles were frequent
(>0.1) in BS-811 (0.302), PB-41 (0.270), PB-49 (0.141),
PB-29 (0.127), PBC-1 (0.126) and BS-9 (0.101). The summarized genetic
indices (Table 1) reveal a gradual decline in gene diversity (He) and
that the populations were not under the HWE. The highest value of
allelic richness was reached in 2005, just slightly higher than in the
two preceding sampling years, whereas the peak in private allelic
richness was reached in 2007. Nei’s genetic distance and the time
between the sampling periods are significantly correlated (Suppl. Table
A.4). Each run in STRUCTURE resulted in four clusters (Fig. 2C). Cluster
1 (blue in Fig. 2C) was more dominant in 2001 and 2003, cluster 2 (pink)
revealed same levels across all years, and clusters 3 (yellow) and 4
(red) were more dominant as from 2005. BAPS suggested two clusters,
years 2001-2003 and 2005-2018, a construction which is in line with the
STRUCTURE analysis. Pairwise Fst values suggest that samplings from
years 2001 and 2003, are significantly different from years 2007, 2013
and 2018. However, also 2007 and 2013 samplings are significantly
different (p<0.05) (Suppl. Table A.5B). These Fst values
further validate the clustering of 2001 with 2003. There was a weak but
significant overall differentiation of the populations (Dest) and a
slight genetic drift (Fs’) in Shilshole (Table 2). Interestingly, Fs’
was in an order of magnitude lower in the period of 2001 and 2003
(0.004) compared to 2013-2018 (0.039). The lower genetic drift between
years 2001 and 2003 is congruent with the clustering of these two
periods.
Edmonds marina
Four sampling periods have yielded 101 tissue samples. In an additional
sampling session (year 1999), no colony was found on the marina’s hard
bottom shallow substrates. All microsatellite loci were polymorphic,
yet, only a single allele was found in 2003 and 2018 in locus BS-9. Out
of the 55 alleles (Suppl. Table A.3), 17 were private alleles, two
private alleles in year 2003, and 5 private alleles in each of the other
sampling years. Null alleles were frequent in PB-41 (0.351), BS-811
(0.279), PB-29 (0.172), BS-8 (0.166) and PB-49 (0.108). The genetic
indices (Table 1) reveal an increase in allelic richness and gene
diversity from 2003 to 2005, with a peak in allelic richness and private
allelic richness in 2005. There was no correlation between Nei’s genetic
distance and the time between the sampling periods (Suppl. Table A.4).
Pairwise Fst values suggest a significant difference between years
2003/2005, 2003/2018 and 2005/2018 (p<0.05) (Suppl. Table
A.5C). The weak Dest was significant (p=0.006) and the Fs’ moderate
(Table 2). BAPS suggested two clusters, one for year 2003 and the second
for 2005-2018. Nevertheless, the inconsistent number of K suggested by
STRUCTURE proposes a very weak population structure. Two clusters were
finally assigned due to constant, but not always tall Delta K peaks at K
= 2 (Fig. 2D). The BAPS results were confirmed by STRUCTURE, with year
2003 being assigned in a single cluster, whereas the other three
sampling dates failing to group into a specific cluster.
Shelton marina
Shelton’s marina was visited only during three sessions, yielding 78
tissue samples. All microsatellite loci were polymorphic, although in
1999, loci BS-8, BS-9 and PB-41, in 2003 locus BS-9 and in 2018 locus
BS-8 were represented by just a single allele. Forty-six alleles were
recorded, whereof 19 were private, e.g., found in a single sampling date
(Suppl. Table A.3). Most private alleles occurred in 2003 (n=9),
followed by 2018 (n=7) and 1999 (n=3). Null alleles were frequent in
four loci (PB-41, PB-49, BS-811 and PB-29), ranging from 0.220 to 0.268.
The genetic indices (Table 1) reveal peaks of allelic richness and
private allelic richness in 2003. Nei’s genetic distance was not
correlated to the time passed between the sampling events (Suppl. Table
A.4). STRUCTURE suggested three clusters (Fig. 2B), however, no
meaningful trend could be detected. BAPS, on the other hand, grouped the
years 1999 and 2003 as one cluster, and 2018 as the second. The
different populations were weakly (p=0.014) separated according to Dest
(Table 2), and the Fs’ was moderate. Pairwise Fst showed a significant
differentiation between 1999/2018 and 2003/2018 (Suppl. Table A.5D),
which is in line with the clusters suggested by BAPS.
Regional scale - Puget
Sound
Overall analysis
At the regional scale, 104 alleles were recorded during the 19-years
sampling period. Nineteen private alleles were found in Des Moines
(26.8% of local alleles), eight (17.4%) in Shelton, nine (14.5%) in
Shilshole and eight (14.5%) in Edmonds. Analyses for the three most
frequent alleles per locus (Fig. 3) revealed just a single allele in
loci BS-8 and BS-9 (181bp and 194bp, respectively) dominant every year
in each location, while loci BS-811, PB-29 and PBC-1 showed variable
allele frequencies. In Edmonds, the third most frequent allele in locus
BS-811 was highly frequent in 2003 and 2018, but absent in the years
between. Only eight alleles were present in every sampled population,
alleles 186bp and 189bp in BS-2, alleles 152bp and 156bp in PB-29, and
alleles 199bp, 202bp, 205bp and 210bp in PBC-1. Some alleles reflect
site specific distributions. For example, allele 178bp in microsatellite
BS-2 was presented in all Shilshole and Shelton populations, but was
absent from Des Moines and Edmonds. In contrast, allele 192bp in
microsatellite BS-8 was present every sampling period in Des Moines and
Edmonds, but completely absent from Shilshole and Shelton.
The Des Moines populations were almost twice as much differentiated
(Dest) compared to the other three marinas (Table 2), a result further
supported by the population structure Fst, revealing the highest values
for Des Moines. For both indices, the differences among the four
locations (Dest and Fst) were higher in the earlier period (1999-2007)
than between 2013 and 2018. For the inbreeding (Fis) index, only Shelton
revealed remarkable lower values. The overall genetic drift values were
similar in Des Moines, Edmonds and Shelton, and less than half in
Shilshole, while for allelic richness the highest numbers were assigned
to Des Moines (Table 1), followed by Shilshole, Edmonds and Shelton. Des
Moines also showed the highest He, also a measure of the evenness of the
allelic frequencies, similar to the number of effective alleles (Brown
& Weir, 1983). These high levels were followed by Edmonds, Shilshole
and Shelton.
Analyzing all 20 populations simultaneously resulted in four BAPS
individual clusters, where each location was assigned to a separate
cluster (Suppl. Fig. A.1). STRUCTURE, on the other hand, suggested two
clusters (Fig. 4A). The Des Moines samples were mainly assigned to
cluster 1 (yellow in Fig. 4A), whereas Shilshole samples belonged
primarily to cluster 2 (red in Fig. 4A), with Shelton’s and Edmonds’
individuals being mixed. Netstruct suggested three highly significant
clusters (Fig. 4B), with Des Moines belonging mainly to cluster one
(blue in Fig. 4B), Shilshole to cluster two (red in Fig. 4B) and Shelton
to cluster three (green in Fig. 4B), whereas Edmonds seems to be
well-mixed.
Gene flow between sites for the whole period studied was somewhat
restricted (Suppl. Fig. A.2), as Shelton received gene flow just from
Shilshole, and thus emerged as the most isolated site, with only 0.02 of
the genetic material being received from other locations, compared to
0.06 in Edmonds and Shilshole and 0.07 in Des Moines. The strongest gene
flow was from Shilshole to Edmonds (0.031), followed by Des Moines to
Shilshole (0.023). Edmonds and Shelton were not connected at all, while
there was a unidirectional connection from Shelton to Des Moines (0.022)
and a bidirectional exchange with Shilshole. Comparing only the sites
without yearly divisions shows highly significant pairwise Fst for all
pairs (Suppl. Table A.5E).
Yearly analyses
Des Moines and Shilshole exhibited in most sampling dates a similar
number of private alleles and generally the highest numbers (Table 3).
Yet, in 2005, Edmonds had more private alleles than Des Moines and
Shilshole combined. Interestingly, Shelton had much fewer private
alleles than Des Moines in 1999 and all other sites in 2018. On a yearly
basis, pairwise Fst between years for the whole Puget Sound area shows
that all combinations except 2001/2003, 2003/2005 and 2007/2013 are
significantly differentiated (Suppl. Table A.5F).
U.S. west coast scale
The analyses included the Puget Sound sites, and two Californian sites,
Santa Cruz (Reem et al., 2013a) and Moss Landing (Karahan et al., 2016),
involving the five shared microsatellite loci (BS-811, PB-29, PB-41,
PB-49, PBC-1) used for analyses in these locations.
Overall
Allelic richness and expected heterozygosity in the Puget Sound
populations were lower than in the two Californian populations (Table
4). Despite similar allelic richness and expected heterozygosity, Moss
Landing and Santa Cruz populations were clustered to two different
groups (orange and red in Fig. 5A) in STRUCTURE, and all four Puget
Sound populations formed a third cluster (yellow in Fig. 5A). BAPS
created three distinct clusters when considering the four Puget Sound
sites as a single location, and no gene flow between any of the sites
was discernible (Suppl. Fig. A.3). Three highly significant clusters
were selected using Netstruct for the west coast analysis (Fig. 5B).
Strikingly, the Santa Cruz populations clustered together to a single
entity, whereas the Moss Landing populations were mostly associated with
the remote Seattle populations, showing minimal similarities to the
close Santa Cruz cluster.
Separate years
During four sampling years (1999, 2001, 2005, 2007) DNA samples were
taken in at least a single Californian and two Puget Sound sites.
Analyses on each year separately, revealed only a single genetic
connection between California and Puget Sound (year 2007), a substantial
gene flow (0.039) from Des Moines to Moss Landing, whereas Santa Cruz,
Shilshole and Edmonds remained isolated (Fig. 6).
Global scale
For the analyses, we used 10 worldwide sites (Table 4) and five shared
microsatellite loci (BS-811, PB-29, PB-41, PB-49, PBC-1). Locus BS-811
had the highest number of alleles in all locations (20-65; Table 4).
When considering the total number of individuals analyzed, Puget Sound
emerged as the region with the lowest number of alleles per individual
on either locus and on the combined loci. This puts the whole
Mediterranean area (Reem et al., 2017), Moss Landing (Karahan et al.,
2016) and South America (Rachel Ben-Shlomo et al., 2010) as containing
the highest numbers of allele per 100 colonies (N=57-66). The number of
alleles in the Puget Sound was notoriously low (N=16), primarily on
locus PBC-1, presenting just 61.5% of alleles as compared to the second
lowest site, Moss Landing. The European and Mediterranean locations
(without Scandinavia) showed the highest number of alleles in PB-29,
PB-41 and PBC-1, and were also leading among the other two loci. The
average expected heterozygosity among Puget Sound populations was by far
the lowest across the globe, suggesting a limited evenness of the allele
frequencies. Furthermore, the allelic richness was among the lowest in
Puget Sound, whereas the highest values were observed in Israel and
Santa Cruz.
The analysis on the frequent alleles (>0.1 in at least a
single population; Suppl. Table A.6) revealed some disparities between
worldwide sites. For example, all the frequent alleles on locus PB-29 in
the U.S. west coast are under 160bp, while in the other regions at least
one allele was >160bp. In the most polymorphic locus
BS-811, most of the European, South American and New Zealand frequent
alleles are <260bp in contrast to the North American
(<300+bp). Locus PB-41 did not display different patterns
among locations, except for Israel (Paz et al., 2003). While in most
sites the majority of frequent alleles was below 180bp, most alleles in
the Israeli populations were >180bp. For locus PB-49,
allele sizes >240bp were just found in Santa Cruz (Reem et
al., 2013a), the US east coast (Stoner, Ben-Shlomo, Rinkevich, &
Weissman, 2002) and the Mediterranean Sea (Reem et al., 2017), while in
Israel (Paz et al., 2003), Scandinavia (Reem, Mohanty, Katzir, &
Rinkevich, 2013b) and South America (Rachel Ben-Shlomo et al.,
2010), the majority of the frequent allele sizes was
<220bp. Locus PBC-1 contained alleles >210bp
everywhere except in the Puget Sound (this study), South America
(Ben-Shlomo et al., 2010) and New Zealand (Ben-Shlomo et al.,
2001). In the Mediterranean Sea (Reem et al., 2017), the
Atlantic coasts of Europe (Ben-Shlomo, Paz, & Rinkevich, 2006) and in
South America (Ben-Shlomo et al., 2010), the alleles are shorter,
<190bp. In New Zealand (Ben-Shlomo et al., 2001), most PBC-1
alleles were between 190 and 200 bp, while in the Puget Sound (this
study), Moss Landing (Karahan et al., 2016) and Israel (Paz et al.,
2003), sizes of 201-210bp were most commonly observed.