DOI: 10.5061/dryad.w0vt4b8q1
title: Exploration of marine lichenized fungi as bioindicators of coastal ocean pollution in the Boston Harbor Islands National Recreation Area

description: This preliminary exploration of marine lichenized fungi (lichens) as
 bioindicators of water pollution examined the distribution of intertidal
 lichen communities in the Boston Harbor Islands National Recreation Area
 with respect to recorded pollution throughout the harbor. We found
 significant negative associations between pollution measurements and the
 health of the lichen community based on cover and species richness. We
 also observed significant differences in species composition between areas
 of higher pollution and areas of lower pollution, though not enough data
 are available to establish the pollution sensitivity or tolerance of
 individual species. We note that difficulties in the collection and
 identification of marine lichens hamper efforts to use them broadly as
 bioindicators. This study suggests that marine lichens could prove useful
 as bioindicators, but more research is needed to understand the
 differential effects of pollution on individual species as well as to
 establish practical procedures both for quantifying marine lichen
 community health and for widespread bioindication using marine lichens.
 Finally, one species collected during this study, Verrucaria ceuthocarpa,
 represents a first report for the Boston Harbor Islands National
 Recreation Area.
description: We tested potential for the use of marine lichens as
 bioindicators by examining their distribution in the Boston Harbor Islands
 NRA and compared those findings with land use histories and water
 pollution analyses in select locations throughout the park. Six pollution
 types were examined for this study, data for most of which came from the
 recent MWRA <i>Boston Harbor Water Quality Updates</i> (2018).
 The data we used included measurements of nitrogen concentration,
 chlorophyll-a concentration, total suspended solids, turbidity,
 qualitative classifications of depositional, erosional, and intermediate
 sediment types, and the nature of pollution of the Weir River Area of
 Critical Environmental Concern (Lefebvre et al. 2002; Pahlevan et al.
 2018; Taylor 2018). Collecting procedures
 were approved by the National Park Service under permit
 #BOHA-2019-SCI-0005. Six areas were sampled based on accessibility and
 distance from historical or current point sources of pollution in the
 Boston Harbor: Bumpkin Island, Grape Island, Lovells Island,
 Peddocks Island, Thompson Island, and World’s End peninsula. The
 coastline was also examined at Rowes Wharf in Boston. All sampling
 occurred throughout the month of August in 2019. We did not take neap and spring
 tides into account due to difficulties in scheduling travel to the
 islands; however, the range of the height of the tide on sampling days was
 approximately two feet (US Harbors 2020). We sampled along the entire
 length of transects of 1 m width that stretched from the low tide mark to
 the high tide mark based on mean lower-low and mean higher-high water data
 (NOAA 2011). In total, 33 transect sites were surveyed. Different islands
 had different numbers of transects based on the estimated length of
 suitable rocky habitat along the shore, with each estimated 100 meters
 corresponding to one sampling site. Satellite images of the Boston Harbor
 were used to estimate shoreline lengths and to observe possible habitats
 (Terrametrics 2020). Sites were chosen ahead of time
 by randomly selecting an integer between 1 and 5000 and estimating the
 site location by moving that many meters counterclockwise from
 the landing dock on each island and from the
 entrance at World’s End. When selected locations did not have suitable
 habitat for marine lichens, new points were chosen using the same
 method. Specimens were identified through
 microscopy following Taylor (1982). For five specimens that proved
 difficult to verify morphologically, identifications were confirmed using
 deoxyribonucleic acid (DNA) extractions and polymerase chain reaction
 (PCR) amplification of the internal transcribed spacer (ITS) ribosomal DNA
 region. We used the DNeasy PowerPlant Pro Kit (Qiagen, Valencia, CA) for
 DNA isolation following the manufacturer’s protocols. Amplification of the ITS was done
 in 25 μL reactions with 12.5 μL of Promega 2× PCR Master Mix
 (Promega Co., Madison, WI), 1.25 μL of forward and reverse 10 μM primer,
 9.0 μL of H<sub>2</sub>O, and 1.0 μL of template DNA.
 Different primer combinations were tested for optimal results, including
 ITS1F (Gardes and Bruns 1993) and ITS9mun (Egger 1995) as forward primers,
 and ITS4 (White et al. 1990) and ITS4A (Larena et al. 1999) as reverse
 primers. Thermocycler conditions were as follows: initial denaturation at
 94 °C for 3:00 min; 35 cycles of denaturation at 94 °C for 1:00 min,
 annealing at 50 °C for 0:45 min, and extension at 72 °C for 1:30 min; and
 final extension at 72 °C for 10:00 min (Haelewaters et al. 2018).
 Purification and Sanger sequencing were outsourced to Genewiz (South
 Plainfield, NJ). Forward and reverse reads were assembled and edited in
 Sequencher 5.2.3 software (Gene Codes Co., Ann Arbor, MI), and final
 sequences were submitted to the National Center for Biotechnology
 Information  GenBank sequence database (accession numbers
 MT809480–MT809484). Collections verified with sequences are specified in
 Table 1. Finally, we compared our samples with voucher specimens from
 LaGreca et al. (2005). Nomenclature follows Index Fungorum (2020).
 Specimens are preserved at FH (Farlow Herbarium, Harvard University,
 Cambridge, MA). Note that field identifications on Bumpkin Island were
 made using Taylor (1982) and comparison with herbarium specimens from
 other islands. No voucher specimens were collected from this
 island  due to practical limitations.
   We measured species richness (i.e., the
 number of species) in each transect and total coverage, size, number of
 thalli, and reproductive potential (density of perithecia) for each
 species. Size, cover, and count of lichen thalli within each sampling site
 were calculated with a combination of measurement in the field (i.e.,
 counts, measurements, and field identifications) and analysis of
 photographs taken at each site. Size was measured by the longest diameter
 of each thallus, and cover was estimated by placing a grid on the
 photograph of each transect and using field notes and pictures to estimate
 percent cover of the transect. We measured perithecial density with
 collected specimens by finding the average number of perithecia within
 three grid templates of 1 cm<sup>2</sup> placed on each
 collection using a dissecting Because our study design
 and data violated conditions for most parametric statistical analyses
 (according to the Anderson-Darling test and D’Agostino
 K<sup>2</sup> tests for normality), we used nonparametric
 models and significance tests to analyze our data. For all significance
 tests, ɑ = 0.05 unless otherwise specified or Bonferroni corrected. We
 used Spearman’s Rho and associated two-tailed significance tests to assess
 nonparametric correlation (MacFarland and Yates 2016), with the following
 hypotheses: H<sub>0</sub>: ρ = 0, H<sub>a</sub>: ρ
 ≠ 0. To compare species richness among different areas of differing levels
 of pollution, we used the Mann-Whitney U test (pairwise) (MacFarland and
 Yates 2016) and Bonferroni-corrected significance tests with hypotheses
 H<sub>0</sub>: η<sub>1</sub> =
 η<sub>2</sub>, H<sub>a</sub>:
 η<sub>1</sub> ≠ η<sub>2</sub>. To show differences
 in species composition among locations of different levels of pollution,
 we used non-metric multidimensional scaling (NMDS) using the Bray-Curtis
 dissimilarity index (McCune and Grace 2002). We used analysis of
 similarities to test for significant differences between species
 composition of different groups, again using Bray-Curtis dissimilarity and
 with the following hypotheses: H<sub>0</sub>: there is no
 difference between the means of two or more groups of (ranked)
 dissimilarities, and H<sub>a</sub>: there is a difference
 between the means of two or more groups of (ranked) dissimilarities.
 Finally, to analyze the main contributors to differences in species
 composition we used the similarity percentage analysis (SIMPER). All tests
 and models were calculated with the ‘vegan’ package in the R language and
 environment for statistical computing (Oksanen et al. 2019; R Core Team,
 Vienna, Austria), with the exception of the pairwise ANOSIM, for which we
 used PAST software (Oslo, Norway), and SIMPER, for which we used PRIMER-7
 (Quest Research Limited, Auckland, New Zealand).            
description: Two README files are attached along with the raw data:
 README.lichens_as_marine_bioindicators_metadata.xlsx details the
 definitions and units for the column headers in the raw data
 files
 README.lichens_as_marine_bioindicators_analysis_procedures.docx
 details the code and procedures for the data analysis in the paper
 publication relating to these data (https://doi.org/10.3119/20)