DOI: 10.6076/d1c884
title: Genetic diversity of Shaw's agave and soil associated microbes in Southern California preserve

description: Shaw’s Agave (Agave shawii ssp. shawii) is an endangered maritime
 succulent growing along the coast of California and Northern Baja
 California. The population inhabiting Point Loma Peninsula has a
 complicated history of transplantation without documentation. The low
 effective population size in California prompted agave transplanting from
 the U.S. Naval base site (NB) to Cabrillo National Monument (CNM). Since
 2008, there are no agave sprouts identified on the CNM site, and concerns
 have been raised about the genetic diversity of this population. We
 sequenced two barcoding loci, rbcL and matK, of 27 individual plants from
 5 geographically distinct populations, including 12 individuals from
 California (NB and CNM). Phylogenetic analysis revealed the three US and
 two Mexican agave populations are closely related and have similar genetic
 variation at the two barcoding regions, suggesting the Point Loma agave
 population is not clonal. Agave-associated soil microbes used
 significantly more carbon sources in CNM soil samples than in NB soil
 likely due to higher pH and moisture content; meanwhile, soil type and
 soil chemistry analysis including phosphorus, nitrate nitrogen, organic
 matter, and metals revealed significant correlations between microbial
 diversity and base saturation (p &lt; 0.05, r2 = 0.3676), lime buffer
 capacity (p &lt; 0.01, r2 = 0.7055), equilibrium lime buffer capacity
 (p &lt; 0.01, r2 = 0.7142), and zinc (p &lt; 0.01, r2 = 0.7136).
 Soil microbiome analysis within the CNM population revealed overall
 expected richness (H’ = 5.647 to 6.982) for Agave species, while the
 diversity range (1 - D = 0.003392 to 0.014108) suggests relatively low
 diversity marked by high individual variation. The most prominent
 remaining US population of this rare species is not clonal and does not
 seem to be threatened by a lack of genetic and microbial diversity. These
 results prompt further efforts to investigate factors affecting
 Agave's reproduction and fitness.
description: <b>Sampling</b> Shaw’s Agave<i>
 </i>leaf tissues were collected from 27 individual plants (Figure
 1G, 1H) across the habitat range (Figure 1G) i.e. Point Loma (CNM, NB),
 Border Field State Park, Rosarito, and Arroyo Hondo. An individual is one
 tightly grouped cluster of rosettes (Figure 1A-C) or a lone rosette
 (Figure 1D). Individual’s samples were collected from three sites in
 California, USA: CNM, CA (7; June 2017); NB, CA (5; June 2017); BC, CA (5;
 March 2008), and two sites in Mexico: AH, MX (5; October 2008); RS, MX (5;
 June 2008). Samples were collected from U.S. National Park Service
 (NPS)-predetermined sites at CNM and NB. Leaf tissue surfaces were cleaned
 with 70% ethanol and excised with sterile scissors. Three replicates from
 each individual’s rosette (Figure 1H) were collected at CNM and NB, placed
 on ice, and stored at -80°C. Leaf tissues collected from BC, AH, and RS
 (Figure 1G) were stored in silica gel. Soil samples were collected at a
 maximal depth of 30 cm within 1-meter from the center of Shaw’s Agave
 rosettes following the root system lines and within 5 cm of peripheral
 roots at CNM and NB using ethanol-treated aluminum coring devices. The
 rhizosphere was not sampled to avoid disturbing the shallow root system of
 these endangered plants. Three soil replicates per agave cluster were
 obtained within 2 meters of each other and placed on ice prior to storage
 at -20°C. <b>Barcoding</b> Approximately 100 mg of Shaw’s
 Agave spineless leaf tissue were snap-frozen with liquid nitrogen and
 homogenized using a mortar and pestle. DNA was isolated using a DNeasy 96
 Plant kit (Qiagen DNeasy,
 Valencia, CA, USA) according to manufacturer’s
 instructions.<b> </b>Barcoding regions of
 <i>rbcL</i> and <i>matK</i> were amplified in an
 Eppendorf Mastercycler using GoTaq Green.<b> </b>Primers
 generated in Primer3plus for <i>rbcL</i> and
 <i>matK</i> are <i>rbcL</i>-F
 5’-CTGCGAATTCCCCCTGCTTA-3’, <i>rbcL</i>-R
 5’-GATCGCGTCCCTCATTACGA-3’, <i>matK</i>-F
 5’-CAAAAGAGGTTCGTTGGGCA-3’, and <i>matK</i>-R
 5’-ATTGGCCCAGATCGGCTTAC-3’. PCR products were purified using a PCR
 clean-up kit (LAMDA Biotech, USA), and samples were sequenced by Eton
 Bioscience Inc., San Diego. <b>Phylogenetic tree
 construction</b> DNA sequences and chromatograms for
 <i>rbcL</i> and <i>matK</i> were visualized in
 FinchTV for quality checking. Multiple sequence alignments were performed
 in GUIDANCE2; DNA segments with low confidence and/or ambiguous reads were
 manually removed. Phylogenetic trees were generated in ClustalOmega using
 neighbor-joining and edited in the Interactive Tree of Life (iTOL; Letunic
 & Bork, 2019). <b>16S rRNA Microbial
 Sequencing and Analysis</b> Genomic DNA was extracted from soil
 samples using a Quick-DNA Fecal/Soil Microbe kit (Zymo Research, USA) per
 manufacturer's instructions. Library preparation was performed using
 the NEXTflex™ 16S V4 Amplicon-Seq Kit 2.0. Libraries were quantified with
 a Bioanalyzer (Agilent 2100 and Agilent 2200 TapeStation) and pooled in a
 MiSeq flow cell for 100,000 reads per sample for 36 samples (12 sites,
 Point Loma). 16S rRNA sequences (~7 million) were imported and analyzed
 using Mothur software (version 1.39.5; Kozich et al., 2013) toolset on
 Galaxy (usegalaxy.org; Afgan <i>et al</i>., 2018). Forward and
 reverse paired sequences were aligned and grouped by sample sites.
 Sequences were aligned to the SILVA 132 bacterial reference (Quast
 <i>et al</i>., 2013; Yilmaz <i>et al.</i>, 2014).
 Sequences aligned to region 10357 to 25452 in the SILVA reference were
 selected for further analysis and denoised by the pre.cluster command.
 Chimeric sequences were screened from the sequences by chimera.vsearch
 with default settings and the remaining sequences taxonomically classified
 (classify.seqs) to the SILVA 132 taxonomy reference. Sequences were
 assigned to operational taxonomic units (OTUs) by cluster.split command
 with the cut-off set at 0.03 (97% identity). Alpha diversity was
 calculated by Shannon’s diversity index, Simpson’s diversity index, and
 observed species richness (OSR); beta diversity was determined by
 calculating Bray-Curtis dissimilarity index and visualized as an NMDS
 plot. <b>Statistical Analysis</b>
 A McDonald-Kreitman test
 (http://mkt.uab.es/mkt/mkt.asp) was performed for US populations (CNM, NB, BC) against Mexican populations (RS, AH) for both <i>rbcL</i> and <i>matK</i> gene sequences to determine divergence between the US and MX populations. Sequence alignments generated in Clustal Omega were used to calculate dS (synonymous substitution) and dN (nonsynonymous substitution) across all samples and between US and MX Shaw’s Agave samples per locus in SNAP v2.1.1. pH and moisture content variation per site were analyzed <i>via</i> one-way ANOVA with post-hoc Bonferroni correction for multiple comparison testing at p &lt; 0.001. t-tests assuming unequal variances were performed to compare richness (S) and evenness (E) of microbial activity between sites. AMOVA (analysis of molecular variance) and HOMOVA (homogeneity of molecular variance) were calculated between different geographic clusters of Shaw’s Agave for barcoding and 16S rRNA data. Pearson’s linear correlation coefficients were generated between all soil-related indices and measurements.