Marraffini, m invasibility of communities supplementary Materials

Marraffini, M

INVASIBILITY OF COMMUNITIES

Supplementary Materials



Figure SM1. Experimental Setup. A. attachment of PVC frame that held experimental blocks attached to floating dock. B. View of diver (Heather Hawk) assisting with installation of a block. C. View of a tile several weeks after installation with solitary tunicates and Sabellidae tube worms that were attached using marine epoxy. D. Close up of treatment tiles in in block B showing that attachment of animals using marine epoxy. Photos A, B, D by Scott Gabara; photo C by Michelle Marraffini.

Figure SM2. Total number of recruits after 100 days plotted as a function of original species richness (A); original percent cover (B), and original percent of native species (C). Data are binned across other factors to examine the main effect in each panel.

Table SM1 Approximate sizes of adult organisms used in the experiment. Size is taken as the longest axis of a solitary animal and the diameter of a colonial animal. For anemones, this is their width in their non-contracted form.

Species	Phyla	Body Plan	Range of sizes (cm)
Botrylloides violaceus (I)	Chordata	Colonial	5-10
Watersipora subtorquata (I)	Bryozoa	Colonial	5-10
Mytilus galloprovincialis (I)	Mollusca	Solitary	7-10
Bugula neritina (I)	Bryozoa	Colonial	5-8
Ciona savignyi (I)	Chordata	Solitary	4-8
Diplosoma listerianum (I)	Chordata	Colonial	3-7
Botryllus "schlosseri"(I)	Chordata	Colonial	2-6
Ascidia ceratodes (N)	Chordata	Solitary	7-10
Balanus crenatus (N)	Arthropoda	Solitary	1-2
Corynactis californica (N)	Cnidaria	Solitary	1-2
Metridium senile (N)	Cnidaria	Solitary	3-6
Aplidium californium (N)	Chordata	Colonial	5-10
Distaplia occidentalis (N)	Chordata	Colonial	3-7
Barentsia ramosa (N)	Entoprocta	Colonial	2-6
Eudistylia polymorpha (N)	Annelida	Solitary	10-20
Mytilus californianus (N)	Mollusca	Solitary	7-10

Organisms_recruited_during_the_experiment.'>Table SM2. Organisms recruited during the experiment. Listed in order of appearance and asterisks indicate species that were not originally used in treatments.

Organism			Date of Appearance		Successful Recruit
Phyla	Lowest Taxonomic Level	Native Status	DOY	Day of Experiment	Size (mm)
Annelida	Spirorbis sp.*	Native	7/16/12	14	N/A
Chordata	Diplosoma listerianum	NIS	7/16/12	14	2
Chordata	Ascidiacea (colonial)*	Unknown	7/16/12	14	2
Chordata	Botrylloides violaceus	NIS	7/30/12	28	2
Bryozoa	Watersipora subtorquata	NIS	7/30/12	28	1
Chordata	Botryllus schlosseri	NIS	8/13/12	42	2
Chordata	Ciona savignyi	NIS	8/13/12	42	2
Annelida	Salmacina tribranchiata*	Native	8/13/12	42	2
Arthropoda	Balanus crenatus	Native	9/10/12	70	2
Bryozoa	Bugula neritina*	NIS	9/24/12	84	1
Annelida	Serpula columbiana*	Native	9/24/12	84	2

Table S3. Estimates of variation explained by the GLMMs used in this experiment. ICC refers to interclass correlation, explaining the proportion of variance explained by random intercept over the variance of the residuals, interpreted as the total variance between groups (Zuur et al., 2009). Time is in days into the experiment.

Model	Table	Time	Response	Components	ICC	Rc^2	Rm^2
Overall GLMM	4	100	Recruits	R, PC, PNS, R:PC, Time, Block	0.5	0.85	0.46
Early Recruitment	S2	57	Recruits	R, PC, PNS, R:PC, Time, Block	0.5	0.66	0.32
Stachowicz Comparison	7	71	Recruits	R, PC, PNS, R:PC, Time, Block	0.5	0.66	0.31
Invasions	S3	30	NIS Recruits	R, PC, PNS, R:PC, Time, Block	0.5	0.61	0.21

Table SM4. Estimated parameters from GLMM for the first 57 days. Model for cumulative recruitment during the first 57 days of the experiment.

	Parameter	Estimate	Std. Error	P value	Variance
Fixed	Intercept	1.0382	0.3650	0.00445	--
	Richness (PR)	0.0435	0.0066	3.44e-07	--
	Percent Cover (PC)	0.0118	0.0006	<2e-16
	Percent Native Species (PPNS)	-0.018	0.0002	<2e-16	--
	R:PC	0.0003	0.0003	<2e-16
	Time	-0.0029	0.0019	<2e-16	--
Random	Block	--	--	--	6.631e-01
	Time	--	--	--	1.797e-05

Table SM5. Estimated parameters from GLMM for the first 71 days. Model for cumulative recruitment during the first 71 days of the experiment, for comparison to previous experiments (6, 7).

	Parameter	Estimate	Std. Error	P value	Variance
Fixed	Intercept	1.2907	0.3387	0.00014	--
	Richness (PR)	-0.0349	0.0057	1.6e-09	--
	Percent Cover (PC)	-0.0203	0.0006	<2e-16
	Percent Native Species (PPNS)	-0.0034	0.0002	<2e-16	--
	R:PC	-0.0057	0.0002	<2e-16
	Time	0.0223	0.0012	<2e-16	--
Random	Block	--	--	--	5.713e-01
	Time	--	--	--	6.955-06

Table SM6. Estimated parameters from the best-fit GLMM on NIS recruitment. Modeling cumulative recruitment of NIS during the first month of the experiment.

	Parameter	Estimate	Std. Error	P value	Variance
Fixed	Intercept	-0.0585	0.4528	0.897	--
	Richness (PR)	-0.013	0.0218	0.551	--
	Percent Cover (PC)	-0.0256	0.0023	<2e-16
	Percent Native Species (PNS)	-0.004	0.0009	8.39e-06	--
	R:PC	-0.0051	0.0009	4.95e-08
	Time	0.0676	0.0095	1.01e-12	--
Random	Block	--	--	--	0.6764
	Time	--	--	--	0.0003

Genetic Methods

When morphological identification was not feasible, genetic methods were used to confirm the identity of recruited individuals (genus or species) and organisms in community manipulations. A sample of DNA was extracted from fresh tissue using Promega© Wizard 96-well DNA extraction kits following manufacturer’s protocol, with the additional step of tissue maceration by bead beating before overnight incubation. Polymerase chain reaction (PCR) amplified the COI region with primers jgLCO1490 and jgHCO2198 [1x Green Gotaq Master Mix, Promega (Cat #PRM7123), 0.2 M BSA, 1.5 mM MgCl₂, 0.2 μM of each primer] using a three-step thermocycler program (three minute at 94C followed by 30 cycles of 94C for one minute as a denaturation step, 47C for one minute as the annealing temperature, and an extension step at 72C for one minute) using universal COI primers (Geller et al. 2013). PCR products deemed successful as bright single bands visualized by agarose electrophoresis were sequenced by the Sanger method (Elim Biopharmaceuticals, Hayward, CA). Geneious^© software (by Biomatters Ltd.) was used to assemble forward and reverse reads, and edited sequences were compared to known sequences in Genbank (National Center for Biotechnology Information) and a reference library developed in the San Francisco invasive species monitoring program (Geller et al. 2013). Similarity of sequences from unknown specimens to a reference sequence of 95% or greater was considered a match for identification.

Results

Ninety-three samples were successfully amplified by PCR and sent for Sanger sequencing. Of these samples 61 sequences, showed strong quality sequences that could be used for analysis (Table S4). This analysis helped resolve the identification of two species (an anemone and Balanus sp.) too small for morphological identification. These specimens had results from Genbank but the identifications were to species never recorded in this area and the matches were suspect. Comparisons to the Geller database revealed better matches and identifications of species known to inhabit these areas. Several specimens remained unresolved as the samples failed to amplify or to give a clean, high quality sequence. Further work is needed to resolve the identity of these samples. The unresolved species were not assigned native or NIS status and were removed from analyses on invasion success.

References

Geller, J. , Meyer, C., Parker, M. and Hawk, H. 2013. Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Molecular Ecology Resources 13(5) 851-861.

Table S4. Field and molecular identifications of organisms used in treatments. Shows comparisons with Genbank database based on the best sequence.

Field Identification	Number of Samples	Molecular Identification	Accession Number	Percent Identical	Final Identification
Anemone (small brown)	4	Aiptasia pulchella	HG423158.1	95	Unknown
Balanus sp.	5	Amphibalabus reticulatus	JQ035518.1	89	Unknown
Lamillaria sp.	4	Assiminea pecos	DQ533844.1	83	Lamillaria sp.
Botrylloides violaceus	7	Botrylloides violaceus	GQ365691	100	Botrylloides violaceus
Colonial Tunicate	3	Botryllus schlosseri	JN083284	100	Botryllus schlosseri
Botryllus schlosseri	3	Botryllus schlosseri	JN083238	99	Botryllus schlosseri
Bugula sp. (brown)	1	Bugula neritina	KC129832.1	99	Bugula neritina
Bugula sp. (white)	1	Bugula stolonifera	KC129849.1	100	Bugula stolonifera
Encrusting Bryozoa	2	Celleporaria oculata	AY168607.1	94	Unknown
Corynactis californica	1	Corynactis californica	AB441257.1	100	Corynactis californica
Sabellid sp.	3	Eudistylia vancouveri	HM473366.1	90	Eudistylia polymorpha
Sabellid sp. (smaller tube)	2	Megalomma splendida	HM473463	98	Megalomma splendida
Mytilus galloprovincalis	6	Mytilus galloprovincalis	GQ480284.1	100	Mytilus galloprovincalis
Sponge (yellow)	3	Haliclona xena	JN242209.1	99	Haliclona sp.
Anemone (small)	5	Metridium senile	HG423143	99	Metridium senile
Mytilus californianus	4	Mytilus californianus	GQ902188.1	99	Mytilus californianus
Watersipora sp.	2	Watersipora sutorquata	DQ417456.1	99	Watersipora sutorquata
Tube (Amphipod)	1	Ericthonius brasiliensis	JX545463	89	Amphipoda
Diplosoma listerianum	4	Diplosoma listerianum	KF791868.1	99	Diplosoma listerianum
Total	61

Table S5. Field and molecular identifications of organisms unresolved by Genbank. Shows comparisons with Genbank database and Geller lab database based on the best sequence.

Field Identification	Genbank Identification	Accession Number	Percent Identical	Geller Lab Identification	Percent Identification	Finial Identification
Anemone (small brown)	Aiptasia pulchella	HG423158.1	95	Diadumene lineata	100	Diadumene lineata
Balanus sp.	Amphibalabus reticulatus	JQ035518.1	89	Balanus crenatus (haplotype A)	98	Balanus crenatus