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Characterisation of polymorphic microsatellite markers in the widespread Australian seagrass, Posidonia Australis Hook. F. (Posidoniaceae), with cross-amplification in the sympatric P. Sinuosa

Authors:
Elizabeth A. Sinclair at University of Western Australia
Elizabeth A. Sinclair
Janet Anthony at Government of Western Australia
Janet Anthony
Grey Coupland at Murdoch University
Grey Coupland
Michelle Waycott at University of Adelaide
Michelle Waycott
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Abstract and Figures

We developed 10 polymorphic microsatellite markers in the Australian seagrass Posidonia australis Hook. f. Markers were screened for their ability to detect within- and among-population genetic structure and variation. The markers showed a range in levels of polymorphism from fixed differences between the two sampled seagrass meadows to high levels of heterozygosity. These markers will be used to estimate gene flow across the species range, characterise the mating system through paternity analysis and pollen dispersal, characterise the nature and extent of clonality, and determine the genetic differentiation of local seagrass meadows to provide information on where to source local genetic provenance material for seagrass restoration projects. Seven of the 10 loci also amplified in the sympatric P.sinuosa and will be useful in future studies in population genetics and hybridisation.
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TECHNICAL NOTE
Characterisation of polymorphic microsatellite markers
in the widespread Australian seagrass, Posidonia australis
Hook. f. (Posidoniaceae), with cross-amplification
in the sympatric P. sinuosa
E. A. Sinclair ÆJ. Anthony ÆG. T. Coupland ÆM. Waycott ÆM. D. Barrett Æ
R. L. Barrett ÆM. L. Cambridge ÆM. J. Wallace ÆK. W. Dixon Æ
S. L. Krauss ÆG. A. Kendrick
Received: 17 July 2009 / Accepted: 31 July 2009 / Published online: 26 August 2009
ÓSpringer Science+Business Media B.V. 2009
Abstract We developed 10 polymorphic microsatellite
markers in the Australian seagrass Posidonia australis
Hook. f. Markers were screened for their ability to detect
within- and among-population genetic structure and vari-
ation. The markers showed a range in levels of polymor-
phism from fixed differences between the two sampled
seagrass meadows to high levels of heterozygosity. These
markers will be used to estimate gene flow across the
species range, characterise the mating system through
paternity analysis and pollen dispersal, characterise the
nature and extent of clonality, and determine the genetic
differentiation of local seagrass meadows to provide
information on where to source local genetic provenance
material for seagrass restoration projects. Seven of the 10
loci also amplified in the sympatric P. sinuosa and will be
useful in future studies in population genetics and
hybridisation.
Keywords Posidonia australis Microsatellites
Clonal diversity Restoration Cross-species
amplification Posidonia sinuosa
Seagrasses are important to the health and productivity of
temperate and tropical coastal marine environments and
occur in greatest abundance in sheltered coastal embay-
ments and estuaries (Orth et al. 2006). Significant decline
in seagrass communities has been recorded in many parts
of the world (Walker and McComb 1992; Kendrick et al.
2002; Waycott et al. 2009) and this loss has been correlated
with human development and eutrophication (Orth et al.
2006). Aside from the obvious long-term ecological
impacts, there is a very real and large commercial loss to
the fishing industry associated with seagrass decline
(Costanza et al. 1997; Bjork et al. 2008). The restoration of
seagrass meadows following their disturbance or removal
from coastal marine environments is a global priority
(Fonseca et al.2000), with research focused on developing
methods of transplanting vegetative units (sprigs, plugs,
sods) and seedlings (e.g., Kirkman 1998; Paling et al. 2001;
Bastyan and Cambridge 2008).
Seagrasses are angiosperms, producing pollen and seeds
that disperse through the water column as well as spreading
vegetatively through rhizome extension (Edgar 2000). An
understanding of the extent of seed and pollen dispersal,
genetic composition of individual meadows, within and
among population genetic structure, and the mating system,
will have important implications for sourcing propagules
for restoration of damaged meadows. The Australian sea-
grass Posidonia australis Hook. f. is widespread along the
Australian coastline, occurring from Shark Bay in northern
Western Australia around the southern coast to Lake
Macquarie in central New South Wales, and along the
northern coast of Tasmania (Gobert et al. 2006). Population
differentiation at a broad geographic scale has been
reported in P. australis, but these results were based on a
low number of weakly polymorphic markers e.g., five
polymorphic allozyme loci (Waycott et al. 1997) and 28
E. A. Sinclair (&)J. Anthony M. D. Barrett
R. L. Barrett M. J. Wallace K. W. Dixon S. L. Krauss
Botanic Gardens & Parks Authority, Fraser Avenue,
West Perth, WA 6005, Australia
e-mail: esinclair@iinet.net.au
E. A. Sinclair G. T. Coupland M. D. Barrett
R. L. Barrett M. L. Cambridge M. J. Wallace
K. W. Dixon S. L. Krauss G. A. Kendrick
School of Plant Biology, University of Western Australia,
Crawley, WA 6009, Australia
M. Waycott
School of Marine and Tropical Biology, James Cook University,
Townsville, QLD 4811, Australia
123
Conservation Genet Resour (2009) 1:273–276
DOI 10.1007/s12686-009-9067-y
Table 1 Characteristics of 10 polymorphic microsatellite loci isolated from Posidonia australis
Locus ID Repeat motif Primer sequence (50–30) Dye
label
Product
length
Anneal
temp (°C)
Mg
conc.
Size
range (bp)
No.
alleles
Genbank
accession number
PaA1-F (TG)16 CCA CCA ATG AAA CCA TAA GG D4 240 59 1.5 242–256 8 GQ397100
PaA1-R CTT TGC TGG GAA ATC TAA CG
PaA105-F (AC)15 GCC TCA TCT AAG GAA TCT TTT G D2 201 58 2.0 200–237 8 GQ397101
PaA105-R TCC TAG AAG GGC ATC CTA GAC
PaA120-F (CA)37 TGC CAC TAT GAT TTA TCT CCC D2 205 59 1.75 172–198 8 GQ397102
PaA120-R ATT TCG GTC TAC CTA TTC AAG C
PaB6-F (TG)9 CCC AAC AAT TTG AAG AAA ACA C D4 243 57 2.0 203–245 3 GQ397103
PaB6-R AGC CAA TAG GAC AGA ACT CTT G
PaB8-F (GA)23 CCA AAG AGT GTA GGA AGA GTG C D3 212 60 2.0 188–340 8 GQ397104
PaB8-R TAT TCC AGG CAT GTA ATG TCA G
PaB112-F (CT)16 GCC CTC TCT TAC TCT CTC TCG D4 179 61 2.0 184–190 5 GQ397105
PaB112-R GGA AGT GTT GGA CAG TGA ATC
PaD12-F (TGA)2C(TGA)5 TGA TTC TCG CTT CCT TAC TTA C D3 278 57 2.0 269–288 2 GQ397106
PaD12-R AGG GAC AAC TCT CTC ACC A
PaD113-F (CAT)11 CCT GAT ACC TGC TGT GAT ACC D3 198 60 2.0 189–207 5 GQ397107
PaD113-R GCA ACT CCT CGT TAC AAT AGC
PaD114-F (GA)8 (GT)7 CAC CAC GAA CAA AAG TCA TAC C D2 228 57 2.0 209–245 2 GQ397108
PaD114-R TAT TGA GAT CGG GTG ATG TGA T
Pa118/9F (CT)12 GAA GAC CAG ATG TTG ACA D2 125 48 2.0 125–143 6 GQ397109
Pa118/9R ATT TCA GAC TGC TGG GCC
274 Conservation Genet Resour (2009) 1:273–276
123
polymorphic RAPD DNA bands (Waycott 1998). Allo-
zyme markers indicated significant differentiation between
western and eastern Australian populations, while RAPDs
indicated a pattern of isolation by distance. However, the
pattern in allozymes was driven largely by a single locus.
Here, we present a suite of polymorphic microsatellite
DNA markers for P. australis to improve the resolution of
population genetic structure.
DNA was extracted using a PVP-SDS method (Waycott
and Barnes 2001) from fresh frozen leaf material collected
from Cockburn Sound, Western Australia. A microsatellite
repeat enriched DNA library was constructed using the
Hamilton linker system to prevent and identify concate-
nated genomic fragments in the final products (Hamilton
et al. 1999). Genomic library enrichment protocols,
screening, and primer design were conducted according to
Fox et al. (2007) using 5 lg total genomic DNA extracted
using a Qiagen Plant DNA extraction kit. A total of
424 clones were screened; 32 contained repeated DNA
sequences and primers were synthesized for 14 of these.
DNA was also sent to Genetic Identification Services Inc.
(http://www.genetic-id-services.com/) for the development
of four enriched microsatellite libraries: CA, GA, AAC,
and ATG. A total of 71 microsatellite-containing clones
were identified. Primers were designed for 56 of these,
using DesignerPCR version 1.03 (Research Genetics, Inc.).
We optimised and screened a total of 34 loci for variation
using seven samples from seven sites across the complete
geographic range of P. australis. Ten loci were polymor-
phic and are reported here.
Variation was assessed in these 10 loci using samples
arbitrarily collected from two P. australis meadows,
Woodman Point, Cockburn Sound (n=20), Western
Australia and from Port Stephens (n=20), New South
Wales. Microsatellite loci were amplified in 10 ll reaction
volumes containing 2.0 ll59PCR buffer containing
dNTPs (Fisher Biotec), 1.5–2.0 mM MgCl
2
[25 mM],
0.44U Taq (Fisher Biotec), 0.8 ll [1.5–2.0 mM] of each
primer, 2.0 ll of diluted template DNA [5 ng/ll], and
water to 10 ll. The thermal cycling profile consisted of
3 min at 94°C, followed by 30 cycles of 40 s denaturation
at 94°C, 40 s annealing at 48–61°C (Table 1), and 30 s
extension at 72°C, followed by 15 min final extension at
72°C. Improved amplification was obtained using 35
cycles and a 7 min final extension for PaA1, PaB6, and
PaD114. The forward primer from each pair was fluores-
cently end-labeled (Beckman Coulter dyes; Table 1).
Cycling was performed in an MJ 9700 thermal cycler
(Bio-RAD). Multiple PCR products were combined where
possible (pre-determined by size and label) and run on a
CEQ 8800 Genetic Analysis System (Beckman Coulter).
Allele sizes were determined using size standard 400 and
scored using the Beckman Coulter software.
Table 2 Allelic diversity and heterozygosity in P. australis at two different sampling locations, and cross amplification in P. sinuosa
Locus ID Woodman Point, Western Australia Port Stephens, New South Wales P. sinuosa, Western Australia
N Size range
(bp)
No. alleles Obs. Het. Exp.
Het.
N Size range
(bp)
No.
alleles
Obs. Het. Exp. Het. N Size range
(bp)
No.
alleles
Obs. Het. Exp. Het.
PaA1 20 232–275 6 0.550 0.455 20 254–256 2 0.100 0.095 6 241–245 3 0.333 0.486
PaA105 20 192–243 7 0.850 0.783 20 204 1 0.000 0.000 6 –
PaA120 20 184–208 7 0.550 0.573 20 172 1 0.000 0.000 6 –
PaB6 20 243–245 2 0.550 0.439 15 238 1 0.000 0.000 6 245–247 2 0.500 0.375
PaB8 20 200–216 6 0.550 0.624 20 216–340 3 0.250 0.224 6 315–331 5 0.833 0.722
PaB112 20 181–189 5 0.500 0.518 20 187 1 0.000 0.000 6 181–197 5 0.833 0.694
PaD12 16 288 1 0.000 0.000 20 269 1 0.000 0.000 6 –
PaD113 20 192–207 4 0.400 0.344 20 189 1 0.000 0.000 6 191 1 0.000 0.000
PaD114 19 209–233 3 0.368 0.314 20 233–245 2 1.000 0.500 5 228–236 3 0.600 0.460
Pa118/9 20 125–143 5 0.550 0.505 20 131–141 2 0.050 0.049 6 135–147 3 0.500 0.486
Conservation Genet Resour (2009) 1:273–276 275
123
The 10 microsatellite loci were also screened using six
samples from the sympatric species, P. sinuosa, collected
from Cockburn Sound and up to 25 km north.PCR con-
ditions were the same as those used for P. australis, that is,
no re-optimisation was carried out. Our immediate interest
in cross-species amplification was to determine the utility
of these markers for the detection of hybridisation.
For the 10 polymorphic microsatellite loci, between 2 and
8 alleles/locus were amplified in P. australis (Table 2).
Some loci showed no variation within meadows, (and fixed
differences between meadows), some were polymorphic for
different alleles across meadows (no sharing of alleles),
and four loci shared four alleles across meadows (PaB8-216,
PaB112-187, PaD114-233, Pa118/9-141). Levels of diver-
sity (allelic diversity, heterozygosity, number of multilocus
genotypes) varied greatly between meadows. There were
only five unique multilocus genotypes in the Port Stephens
meadow while all 20 individuals from Woodman Point had
unique multilocus genotypes. Population data were tested
for deviations from Hardy–Weinberg equilibrium (HWE)
and for linkage disequilibrium using GENEPOP 3.4
(http://genepop.curtin.edu.au/; Raymond and Rousset 1995)
for the Woodman Point population only. All loci were in
HWE and there was no evidence of linkage disequilibrium.
It was not possible to do most tests for the Port Stephens
sample due to low allelic diversity. The high level of
diversity at Woodman Point implies a high degree of out-
crossing in P. australis, however, low numbers of genotypes
at Port Stephens suggests extensive clonality is also a strong
feature within the species, (similar to P. oceanica, Procac-
cini et al. 2002). Locus PaB6 and PaB8 did not amplify
(consistently) well in samples from Port Stephens, and
less stringent PCR conditions were required. These results
suggest deeply divergent eastern and western lineages
within P. australis, consistent with earlier allozyme results
(Waycott et al. 1997).Seven of the 10 loci amplified in
P. sinuosa samples, six of which were polymorphic
(Table 2). These markers may be useful for species identi-
fication and hybrid detection.
The polymorphic microsatellite markers characterised
here will enable an extensive examination of population
genetic structure, mating system, clonality, and dispersal of
pollen and seed within and among seagrass meadows.
Ultimately, these investigations will make a significant
contribution to the generation of genetic guidelines for the
sourcing of propagules for seagrass meadow restoration.
Acknowledgements This project was funded jointly by Cockburn
Cement Ltd and the Western Australian Department of State Devel-
opment (previously Department of Industry and Resources), as part of
the Seagrass Research and Rehabilitation Plan (SRRP). Thanks to
T. Glasby (New South Wales Department of Primary Industries) for
providing samples from Port Stephens, M. Todd (Genetic Identifica-
tion Services), and K. Hillman (Oceanica Consulting, Perth).
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... We ran two levels of experiments: a longer-term two-year experiment focused on N, P and chelated Fe additions to P. australis transplants in Princess Royal and Oyster Harbour and; 2 one-year long experiments comparing transplant and root growth in P. sinuosa and P. australis. The results from the two-year experiment indicated that nitrogen limitation was occurring in Princess Royal Harbour ( Figure 10) whereas in Oyster Harbour, phosphorus was limiting plant growth ( Figure 11) (Cambridge & Kendrick 2009). The addition of chelated Iron (Fe EDTA) produced equivocal results in both Harbours and may be related more to the interactions between the macro-nutrients and the chelator EDTA than with Fe. ...
... The potential for seed and seedling-based restoration was investigated during the Seagrass Research and Rehabilitation Plan (Sinclair et al. 2009, Statton et al. 2013) by University of Western Australian researchers and has continued through Australian Research Council Industry Linkage grants partnering with Cockburn Cement/Adelaide Brighton Pty Ltd (LP100200429, LP130100155, LP130100918, LP160101011). Also, the Seeds for Snapper seed-based community restoration program grew from these initial experimental studies and has been running for 4 years. ...
... Restoration and population genetics of P. australis populations and how this may affect seed and shoot sourcing for restoration (2010-2017)(Sinclair et al. 2009(Sinclair et al. , 2014 • Demographic approaches to seed-based restoration(Ruiz-Montoya et al. 2012, 2015, Statton et al. 2017a).• Seed-based restoration experiments. ...
View
... Total genomic DNA was extracted from frozen shoot meristems using a modified PVP-SDS method [polyvinylpyrrolidone (PVP) and sodium dodecyl sulfate (SDS)] (Sinclair et al., 2009(Sinclair et al., , 2014a. Multilocus genotypes were generated using seven polymorphic microsatellite loci (PaA1, PaA105, PaA120, PaB6, PaB8, PaB112, PaD113) using PCR conditions described by Sinclair et al. (2009). ...
... Total genomic DNA was extracted from frozen shoot meristems using a modified PVP-SDS method [polyvinylpyrrolidone (PVP) and sodium dodecyl sulfate (SDS)] (Sinclair et al., 2009(Sinclair et al., , 2014a. Multilocus genotypes were generated using seven polymorphic microsatellite loci (PaA1, PaA105, PaA120, PaB6, PaB8, PaB112, PaD113) using PCR conditions described by Sinclair et al. (2009). Allele frequencies were calculated using GENODIVE version 2.0b27 (Meirmans and van Tienderen, 2004), which handles genotypes with more than two alleles per locus. ...
... Five microsatellite loci produced three alleles in the UL population (PaA1, PaA105, PaA120, PaB8, PaB112). These loci typically display (homozygous or heterozygous) di-allelic genotypes expected from normally segregating multilocus microsatellite loci (Sinclair et al., 2009(Sinclair et al., , 2014a. However, in the samples from UL, Shark Bay, an additional allele was observed in most samples for between one and three loci (Supplementary Data, Table S1). ...
Reproduction at the extremes: Pseudovivipary, hybridization and genetic mosaicism in Posidonia australis (Posidoniaceae)
Article
Full-text available
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Background and Aims: Organisms occupying the edges of natural geographic ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions often associated with climate change, and exacerbated at species’ geographical range edges, are known to trigger alternative responses in reproduction. We report the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm, Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. We describe these novel plantlets and use a combination of microsatellite DNA markers and Flow Cytometry to determine their origin. Methods: Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of these adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. Key Results: All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. 87% of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2C value) for samples from Woodman Point and Useless Loop was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). Conclusions: Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. We document a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggest that the lower than usual water temperature triggered unfertilized inflorescences to ‘switch’ to growing plantlets that were adventitious clones of their maternal parent. This may have important long term implications as both genetic and ecological constraints may limit the ability to adapt or range shift; the meadow already has low genetic diversity, no sexual reproduction and no seedling recruitment.
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... DNA was extracted from shoot meristem and embryos using methods previously described (Sinclair et al. 2009(Sinclair et al. , 2014a. Seven polymorphic microsatellite loci were used to generate multilocus genotypes (MLGs) using two multiplex mixes containing 5.2 µL of 2× Multimix and 1.98 µL of 5× Q sol (Type-It Microsatellite PCR kit; Qiagen, Hinden, Germany), 1.0 µL of primer mix (PM) and 2.0 µL of 5-10 ng DNA in a 10 µL reaction. ...
... Replicate PCRs were performed to ensure the accuracy of the final data set. There was no evidence of linkage disequilibrium or null alleles at these loci based on previously obtained diploid genotypes (Sinclair et al. 2009(Sinclair et al. , 2014a(Sinclair et al. , b, 2016a; however, we ran all new diploid data (see below) through Micro-Checker v2.2.3 to assess for the presence of scoring error due to stuttering, large allele dropout or null alleles (van Oosterhout et al. 2004; http://www.nrp.ac.uk/nrp-strategic-alliances/elsa/ software/microchecker/). ...
Variation in reproductive effort, genetic diversity and mating systems across Posidonia australis seagrass meadows in Western Australia
Article
Full-text available
  • Aug 2020
Populations at the edges of their geographical range tend to have lower genetic diversity, smaller effective population sizes and limited connectivity relative to centre of range populations. Range edge populations are also likely to be better adapted to more extreme conditions for future survival and resilience in warming environments. However, they may also be most at risk of extinction from changing climate. We compare reproductive and genetic data of the temperate seagrass, Posidonia australis on the west coast of Australia. Measures of reproductive effort (flowering and fruit production and seed to ovule ratios) and estimates of genetic diversity and mating patterns (nuclear microsatellite DNA loci) were used to assess sexual reproduction in northern range edge (low latitude, elevated salinities, Shark Bay World Heritage Site) and centre of range (mid-latitude, oceanic salinity, Perth metropolitan waters) meadows in Western Australia. Flower and fruit production were highly variable among meadows and there was no significant relationship between seed to ovule ratio and clonal diversity. However, Shark Bay meadows were two orders of magnitude less fecund than those in Perth metropolitan waters. Shark Bay meadows were characterized by significantly lower levels of genetic diversity and a mixed mating system relative to meadows in Perth metropolitan waters, which had high genetic diversity and a completely outcrossed mating system. The combination of reproductive and genetic data showed overall lower sexual productivity in Shark Bay meadows relative to Perth metropolitan waters. The mixed mating system is likely driven by a combination of local environmental conditions and pollen limitation. These results indicate that seagrass restoration in Shark Bay may benefit from sourcing plant material from multiple reproductive meadows to increase outcrossed pollen availability and seed production for natural recruitment.
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... Abbreviations by sampling location: with an "m" = fruit collected from parent plants for mating system study, "e" = seeds collected from dispersing fruit. Significant deviation from HWE * DNA was extracted from fresh frozen seeds using a modified PVP-SDS method [polyvinylpyrrolidone (PVP) and sodium dodecyl sulphate (SDS)](Sinclair et al., 2009;Sinclair, Gecan et al., 2014), with the following modification: half a seed sample was added to a 2.0-ml screw-cap plastic tube containing two 0.5-mm ceramic beads and then ground using a FastPrep ® -24 Instrument (MP Biomedicals) for 2 × 40 s. The same seven unlinked polymorphic microsatellite loci(PaA1, PaA105, PaA120, PaB6, PaB8, PaB112and PaD113) were amplified as perSinclair et al. (2009), Sinclair, Gecan, et al. (2014 and PCR products were pooled where possible prior to genotyping on a CEQ 8800 Genetic Analysis System (Beckman Coulter). ...
... Significant deviation from HWE * DNA was extracted from fresh frozen seeds using a modified PVP-SDS method [polyvinylpyrrolidone (PVP) and sodium dodecyl sulphate (SDS)](Sinclair et al., 2009;Sinclair, Gecan et al., 2014), with the following modification: half a seed sample was added to a 2.0-ml screw-cap plastic tube containing two 0.5-mm ceramic beads and then ground using a FastPrep ® -24 Instrument (MP Biomedicals) for 2 × 40 s. The same seven unlinked polymorphic microsatellite loci(PaA1, PaA105, PaA120, PaB6, PaB8, PaB112and PaD113) were amplified as perSinclair et al. (2009), Sinclair, Gecan, et al. (2014 and PCR products were pooled where possible prior to genotyping on a CEQ 8800 Genetic Analysis System (Beckman Coulter). Allele sizes were determined using size standard 400 and scored with the aid of Beckman software. ...
Seeds in motion: Genetic assignment and hydrodynamic models demonstrate concordant patterns of seagrass dispersal
Article
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Movement is fundamental to the ecology and evolutionary dynamics within species. Understanding movement through seed dispersal in the marine environment can be difficult due to the high spatial and temporal variability of ocean currents. We employed a mutually enriching approach of population genetic assignment procedures and dispersal predictions from a hydrodynamic model to overcome this difficulty and quantify the movement of dispersing floating fruit of the temperate seagrass Posidonia australis Hook.f. across coastal waters in southwestern Australia. Dispersing fruit cohorts were collected from the water surface over two consecutive years and seeds were genotyped using microsatellite DNA markers. Likelihood‐based genetic assignment tests were used to infer the meadow of origin for seed cohorts and individuals. A three‐dimensional hydrodynamic model was coupled with a particle transport model to simulate the movement of fruit at the water surface. Floating fruit cohorts were mainly assigned genetically to the nearest meadow, but significant genetic differentiation between cohort and most‐likely meadow of origin suggested a mixed origin. This was confirmed by genetic assignment of individual seeds from the same cohort to multiple meadows. The hydrodynamic model predicted 60% of fruit dispersed within 20 km, but that fruit were physically capable of dispersing beyond the study region. Concordance between these two independent measures of dispersal provide insight into the role of physical transport for long distance dispersal (LDD) of fruit and the consequences for spatial genetic structuring of seagrass meadows. This article is protected by copyright. All rights reserved.
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... The outer leaf sheaths of each shoot were removed to expose the white meristem tissue, which was then frozen at −80 ∘ C prior to DNA extraction. Total genomic DNA was extracted from frozen shoot meristems using a modified PVP (polyvinylpyrrolidone)-SDS (sodium dodecyl sulfate) method (Sinclair et al. 2009), with the following modification: samples were ground using a FastPrep-24 Instrument (MP Biomedicals, Seven Hills, Australia; Sinclair et al. 2014). A set of eight polymorphic microsatellite markers (PaA1, PaA105, PaA120, Restoration Ecology PaB8, PaB112, PaD12, PaD113, and Pa118/9) developed for P. australis (Sinclair et al. 2009) were used to check the genotypes of transplant samples. ...
... Total genomic DNA was extracted from frozen shoot meristems using a modified PVP (polyvinylpyrrolidone)-SDS (sodium dodecyl sulfate) method (Sinclair et al. 2009), with the following modification: samples were ground using a FastPrep-24 Instrument (MP Biomedicals, Seven Hills, Australia; Sinclair et al. 2014). A set of eight polymorphic microsatellite markers (PaA1, PaA105, PaA120, Restoration Ecology PaB8, PaB112, PaD12, PaD113, and Pa118/9) developed for P. australis (Sinclair et al. 2009) were used to check the genotypes of transplant samples. All transplants from SG were genotyped (with the exception of 19 samples spread across all treatments that were lost during transportation), as SG was known to have high-genetic diversity. ...
Assessing the effect of genetic diversity on the early establishment of the threatened seagrass Posidonia australis using a reciprocal‐transplant experiment
Article
Full-text available
  • May 2018
  • RESTOR ECOL
Two common goals for restoration are rapid plant establishment and long-term plant persistence. The success of transplanted populations may be jeopardized if the donor transplants are not genetically diverse, and/or poorly matched to their new environment. Here, we test the effects of local adaptation and plot-level genetic diversity on the early establishment phase of a threatened seagrass species, Posidonia australis, by performing a reciprocal transplant experiment across two genetically and geographically distinct populations in southeastern Australia. Posidonia australis is a long-lived, slow-growing species that has no seed bank, and the successful transplantation of live shoots and seedlings is the only available restoration method. Our results show a strong effect of local adaptation and genetic diversity on P. australis survivorship and performance over the first 6 months following transplantation. High-genetic diversity plots displayed higher survival rates and exhibited reduced productivity and increased carbohydrate reserves within the rhizome. This suggests that high-diversity plots included shoots that were conserving energy stores by actively reducing growth rates during the early stages of transplantation. The lowest diversity plots exhibited high leaf and root productivity and corresponding low carbohydrate reserves. This may be a sign of stress in the low-diversity transplants, potentially explaining the very low survival rate. We suggest that future restoration efforts source donor transplants from multiple local sources to ensure both local adaptation and sufficient genetic diversity to increase the likelihood of early establishment success.
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... It could also be argued that the majority of SSR work has arisen from the Americas, Europe and Asia to date (Table 2). Recent efforts in Australia have; however, identified SSRs for Australian seagrass species Z. muelleri, Z. nigricalis, P. australis and P. sinuosa (Sinclair et al. 2009;Sherman et al. 2012;Smith et al. 2013). More work needs to be conducted in the Indo-Pacific, a hot spot for seagrass diversity. ...
... Additionally, the numbers of polymorphic loci remain limited for Halophila. In seagrasses, validated SSRs have shown the capacity to cross-amplify in other closely related species of seagrass (Reusch 2000;Sinclair et al. 2009;Smith et al. 2013); however, crossamplification of markers has not been a major focus in seagrass biology as compared to crop plants. More recently, novel methods have been developed to detect genic SSRs in seagrasses. ...
The emergence of molecular profiling and omics techniques in seagrass biology; furthering our understanding of seagrasses
Article
Full-text available
  • Sep 2016
  • FUNCT INTEGR GENOMIC
Seagrass meadows are disappearing at alarming rates as a result of increasing coastal development and climate change. The emergence of omics and molecular profiling techniques in seagrass research is timely, providing a new opportunity to address such global issues. Whilst these applications have transformed terrestrial plant research, they have only emerged in seagrass research within the past decade; In this time frame we have observed a significant increase in the number of publications in this nascent field, and as of this year the first genome of a seagrass species has been sequenced. In this review, we focus on the development of omics and molecular profiling and the utilization of molecular markers in the field of seagrass biology. We highlight the advances, merits and pitfalls associated with such technology, and importantly we identify and address the knowledge gaps, which to this day prevent us from understanding seagrasses in a holistic manner. By utilizing the powers of omics and molecular profiling technologies in integrated strategies, we will gain a better understanding of how these unique plants function at the molecular level and how they respond to on-going disturbance and climate change events.
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... Thirty individual shoots from each of the 44 P. australis meadows were sampled following methods described by Sinclair et al. (2014). DNA was extracted from meristem tissue and genotyped for nine polymorphic microsatellite loci (PaA1, PaA105, PaA120, PaB6, PaB8, PaB112, PaD12, PaD113, and Pa118/9), using methods previously described (Sinclair et al., 2009. We combined data from published regional studies (Evans et al., 2014;Sinclair et al., 2014Sinclair et al., , 2016 with sampling from an additional 14 meadows for a range-wide assessment. ...
Historic and contemporary biogeographic perspectives on range-wide spatial genetic structure in a widespread seagrass
Article
Full-text available
  • Mar 2023
Historical and contemporary processes drive spatial patterns of genetic diversity. These include climate-driven range shifts and gene flow mediated by biogeographical influences on dispersal. Assessments that integrate these drivers are uncommon, but critical for testing biogeographic hypotheses. Here, we characterize intraspecific genetic diversity and spatial structure across the entire distribution of a temperate seagrass to test marine biogeographic concepts for southern Australia. Predictive modeling was used to contrast the current Posidonia australis distribution to its historical distribution during the Last Glacial Maximum (LGM). Spatial genetic structure was estimated for 44 sampled meadows from across the geographical range of the species using nine microsatellite loci. Historical and contemporary distributions were similar, with the exception of the Bass Strait. Genetic clustering was consistent with the three currently recognized biogeographic provinces and largely consistent with the finer-scale IMCRA bioregions. Discrepancies were found within the Flindersian province and southwest IMCRA bioregion, while two regions of admixture coincided with transitional IMCRA bioregions. Clonal diversity was highly variable but positively associated with latitude. Genetic differentiation among meadows was significantly associated with oceanographic distance. Our approach suggests how shared seascape drivers have influenced the capacity of P. australis to effectively track sea level changes associated with natural climate cycles over millennia, and in particular, the recolonization of meadows across the Continental Shelf following the LGM. Genetic structure associated with IMCRA bioregions reflects the presence of stable biogeographic barriers, such as oceanic upwellings. This study highlights the importance of biogeography to infer the role of historical drivers in shaping extant diversity and structure.
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... Microsatellite markers are generally regarded as neutral, i.e. the loci are not under selection, and hence meet the assumptions of many of the population genetic analyses. The recent and ongoing development of microsatellite DNA markers for many Australian seagrass species will improve our understanding of genetic connectivity processes in marine plants: Posidonia australis (Sinclair et al. 2009); Zostera muelleri (Sherman et al. 2012); Heterozostera nigricaulis (Smith et al. 2013) (for the current status of the genus Heterozostera, see Appendix); Cymodocea rotundata (Arriesgado et al. 2014b); Cymodocea serrulata (Arriesgado et al. 2014a); Thalassia hemprichii (Wainwright et al. 2013b;van Dijk et al. 2014); Halophila ovalis (Xu et al. 2010); Enhalus acoroides (Nakajima et al. 2012), Syringodium isoetifolium (Matsuki et al. 2013;Wainwright et al. 2013a), Halodule uninervis, Ruppia tuberosa and Amphibolis antarctica (van Dijk personal communication). ...
Genetic Connectivity in Tropical and Temperate Australian Seagrass Species: Structure, Ecology and Conservation
Chapter
  • Jul 2018
Connectivity among populations influences resilience, genetic diversity , adaptation and speciation, so understanding this process is fundamental for conservation and management. This chapter summarises the main mechanisms of gene flow within and among seagrass meadows, and what we know about the spatial patterns of gene flow around Australia’s coastline. Today a significant body of research on the demographic and genetic connectivity of Australian seagrass meadows has developed. Most studies have focused on the genera Posidonia, Zostera, Heterozostera and Thalassia, in tropical and temperate systems across a range of habitats. These studies have shown overwhelmingly, that sexual reproduction is important for meadow persistence, as in most cases Australian seagrass meadows are genotypically diverse, with moderate to high levels of genotypic diversity. This high diversity could be generated through demographic connectivity, recruitment of individuals sourced from within a meadow, or from dispersal between meadows. Attempts to understand the relative significance of these processes are limited, highlighting a major gap in our understanding. Genetic structure is apparent across a range of spatial scales, from m’s to 100’s to 1000’s km. At local and regional scales, particularly in confined systems such as estuaries and bays, it is not necessarily the dominant oceanographic currents influencing patterns of genetic connectivity, but local eddies, winds and tides. Over larger spatial scales, isolation by distance is consistently significant, with unique genetic clusters spreading over 100s of kilometres. This indicates that regional structure occurs at the limits of long distance dispersal for the species and this is particularly evident where meadows are highly fragmented. The number of genetic studies on Australian seagrasses has increased dramatically recently; however, there are still many opportunities to improve our understanding through focusing on species with different dispersal potentials, more detailed sampling across a range of spatial and temporal scales and combining ecological and modelling approaches.
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... Collection, handling and laboratory methods are described in Sinclair et al. (2014). Nine polymorphic microsatellite loci (PaA1, PaA105, PaA120, PaB6 PaB8, PaB112, PaD12, PaD113, Pa118/9) were genotyped using methods described in Sinclair et al. (2009Sinclair et al. ( , 2014. We also included data for three meadows from southern New South Wales (Evans et al., 2014). ...
Genetic signatures of Bassian glacial refugia and contemporary connectivity in a marine foundation species
Article
Full-text available
  • Jun 2016
Aim To examine the population genetic structure in Posidonia australis mead- ows, a marine foundation species capable of long distance dispersal (LDD), and the role of historical versus contemporary processes in shaping post Last Glacial Maximum (LGM) re-colonization. Location Southeastern Australia including the Bass Strait Islands. Methods We generated multilocus genotypes and assessed spatial patterns of genetic diversity. Relationships among meadows were assessed in terms of his- torical sea level changes, oceanic boundary currents and contemporary seed dispersal based on a hydrodynamic model. Results There was strong regional spatial genetic structuring among P. aus- tralis meadows in south-eastern Australia, which was congruent with three rec- ognized marine biogeographical provinces [Peronian (eastern), Flindersian (western and southern), and Maugean (south-eastern)]. The genetic data sug- gest Maugean meadows persisted in isolation during the LGM, with evidence for admixture and contemporary gene flow. Simulated dispersal events identi- fied high rates of local and regional demographic connectivity, with evidence for occasional LDD events. Main conclusions The strong regional differentiation is consistent with long- term barriers to dispersal persisting in the marine environment through many sea level fluctuations. Bass Strait Island meadows all have strong signals of genetic admixture. A weak but significant isolation by distance relationship is consistent with a historical signal and contemporary seed dispersal mostly within the Bass Strait.
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... Immediately after collection, shoots were placed on ice and returned to the laboratory. A set of eight polymorphic microsatellite markers (developed by Sinclair et al., 2009) were used to determine levels of genetic diversity within and among the 12 P. australis meadows sampled. Following amplification (see Evans et al., 2014 for full protocol), multiple PCR products were combined where possible (pre-determined by size and label) and run on a CEQ 8800 Genetic Analysis System (Beckman Coulter). ...
Genotypic richness predicts phenotypic variation in an endangered clonal plant
Article
Full-text available
  • Feb 2016
Declines in genetic diversity within a species can affect the stability and functioning of populations. The conservation of genetic diversity is thus a priority, especially for threatened or endangered species. The importance of genetic variation, however, is dependent on the degree to which it translates into phenotypic variation for traits that affect individual performance and ecological processes. This is especially important for predominantly clonal species, as no single clone is likely to maximise all aspects of performance. Here we show that intraspecific genotypic diversity as measured using microsatellites is a strong predictor of phenotypic variation in morphological traits and shoot productivity of the threatened, predominantly clonal seagrass Posidonia australis, on the east coast of Australia. Biomass and surface area variation was most strongly predicted by genotypic richness, while variation in leaf chemistry (phenolics and nitrogen) was unrelated to genotypic richness. Genotypic richness did not predict tissue loss to herbivores or epiphyte load, however we did find that increased herbivore damage was positively correlated with allelic richness. Although there was no clear relationship between higher primary productivity and genotypic richness, variation in shoot productivity within a meadow was significantly greater in more genotypically diverse meadows. The proportion of phenotypic variation explained by environmental conditions varied among different genotypes, and there was generally no variation in phenotypic traits among genotypes present in the same meadows. Our results show that genotypic richness as measured through the use of presumably neutral DNA markers does covary with phenotypic variation in functionally relevant traits such as leaf morphology and shoot productivity. The remarkably long lifespan of individual Posidonia plants suggests that plasticity within genotypes has played an important role in the longevity of the species. However, the strong link between genotypic and phenotypic variation suggests that a range of genotypes is still the best case scenario for adaptation to and recovery from predicted environmental change.
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Pilot Experiments on Planting Seedlings and Small Seagrass Propagules in Western Australia
Article
Full-text available
  • Dec 1999
  • MAR POLLUT BULL
The aim of this study was to investigate characteristics of some south-western Australian seagrasses that might be useful for further investigation with respect to restoration. Although seeds and fruits of seagrass species may be useful for producing propagules for planting, their collection is based on knowing when they become available. Posidonia australis, P. angustifolia and P. coriacea flower profusely annually, but P. sinuosa does not. Once collected, the fruits dehisce in a few days and are ready for planting. It was found that the best way to plant Posidonia seedlings is in clusters in Growool® blocks but the rhizomes do not spread quickly. Single Posidonia seedlings, when planted out, were not successful and neither were naturally colonizing seedlings of Posidonia sinuosa in unexposed unvegetated areas. Successful planting of seedlings and sprigs of Amphibolis in matting needs months of calm weather for them to spread out from their attaching matting which must be very securely attached to the substratum. Little success was achieved in growing Amphibolis. In a search for Halophila ovalis seeds, few were found because of the patchy nature of their fruiting and small size. Germination was slow but 63% germinated within 6 months.
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Genetic structure and distribution of microsatellite population genetic diversity in Posidonia oceanica in the Mediterranean basin
Article
Full-text available
  • Nov 2002
  • B MAR SCI
The population genetic structure within and between 33 Posidonia oceanica meadows, sampled in the Mediterranean basin from Spain to Turkey, was analysed using microsatellite DNA markers. The populations analysed ranged from being uniclonal (in the North Adriatic) to having more than 50% of diverse genotypes. Cluster analysis of (δμ)2 genetic distance showed the existence of three population groups, characterised by the presence of north Tyrrhenian, south Tyrrhenian and eastern Mediterranean populations, respectively. Population groups reflect genetic isolation caused by reduced gene flow related to present and past current regimes and to post-glacial re-colonisation events. Low genetic polymorphism and different scales of population genetic sub-structure are present in P. oceanica and could be significant to the capacity for survival and expansion of the species in the highly impacted coastal Mediterranean environment.
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Genetic variation within and between populations of Posidonia australis, a hydrophilous, clonal seagrass
Article
Full-text available
  • Oct 1997
  • HEREDITY
Allozyme diversity was surveyed at 15 loci across 22 populations of the hydrophilous seagrass Posidonia australis (Hook. f). Substantial genetic variation was detected (HT = 0.311) with a high proportion of this variation partitioned between populations (GST = 0.623). The high value of GST is attributed to large geographical distances between many of the populations and several of the extreme north-western populations having fixed homozygous genotypes. Southwestern populations of P. australis were the most variable and these correlate with the highest species diversity in this genus. Intermediate levels of genetic diversity are observed in P. australis when compared with other hydrophilous angiosperms. Average gene diversity values for hydrophilous taxa surveyed to date indicate lower HT and higher GST values than an average reported for 468 plant taxa. Patterns of genetic variability in different regions of the distribution of P. australis may reflect past evolutionary diversification into novel environments and subsequent dispersal following the rifting of Australia from Antarctica in the early Tertiary.Keywords: allozymes, clonal, evolution, hydrophily, seagrass
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A Global Crisis for Seagrass Ecosystems
Article
Full-text available
  • Dec 2006
Seagrasses, marine flowering plants, have a long evolutionary history but are now challenged with rapid environmental changes as a result of coastal human population pressures. Seagrasses provide key ecological services, including organic carbon production and export, nutrient cycling, sediment stabilization, enhanced biodiversity, and trophic transfers to adjacent habitats in tropical and temperate regions. They also serve as “coastal canaries,” global biological sentinels of increasing anthropogenic influences in coastal ecosystems, with large-scale losses reported worldwide. Multiple stressors, including sediment and nutrient runoff, physical disturbance, invasive species, disease, commercial fishing practices, aquaculture, overgrazing, algal blooms, and global warming, cause seagrass declines at scales of square meters to hundreds of square kilometers. Reported seagrass losses have led to increased awareness of the need for seagrass protection, monitoring, management, and restoration. However, seagrass science, which has rapidly grown, is disconnected from public awareness of seagrasses, which has lagged behind awareness of other coastal ecosystems. There is a critical need for a targeted global conservation effort that includes a reduction of watershed nutrient and sediment inputs to seagrass habitats and a targeted educational program informing regulators and the public of the value of seagrass meadows.
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AFLP diversity within and between populations of the Caribbean seagrass Thalassia testudinum (Hydrocharitaceae)
Article
Full-text available
  • Dec 2001
Genetic variation was assessed in the seagrass Thalassia testudinum from three regions of the Caribbean and north Atlantic using allozyme electrophoresis and amplified fragment length polymorphism (AFLP) analysis. Very low allozyme variability was detected among the 196 shoots analyzed from a range of sites in the San Blas region of Panama. AFLP markers detected high similarity (0.87) among the population samples surveyed from Bermuda and Panama across six AFLP primer pairs and over 260 banding positions. High levels of gene flow were detected between all the sites analyzed (N m>1.7). Significantly complete genotypic similarity was observed between samples from Bermuda and Panama, indicating that long distance vegetative fragment dispersal is highly probable. Very low genetic differentiation between all sites, even Bermuda and Panama, some 2,700 km apart, agrees with other studies and is further evidence of a highly uniform gene pool in T. testudinum. High levels of genetic uniformity in T. testudinum may be related to long-term environmental change over its geographic range. While AFLP analysis proved useful in determining genetic variation in this seagrass, the application of co-dominant markers such as microsatellites will be more informative in determining the nature of genetic uniformity and its adaptive significance in T. testudinum.
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Integrating biology and economics in seagrass restoration: How much is enough and why?
Article
  • Jul 2000
  • ECOL ENG
Although success criteria for seagrass restoration have been in place for some time, there has been little consistency regarding how much habitat should be restored for every unit area lost (the replacement ratio). Extant success criteria focus on persistence, area, and habitat quality (shoot density). These metrics, while conservative, remain largely accepted for the seagrass ecosystem. Computation of the replacement ratio using economic tools has recently been integrated with seagrass restoration and is based on the intrinsic recovery rate of the injured seagrass beds themselves as compared with the efficacy of the restoration itself. In this application, field surveys of injured seagrass beds in the Florida Keys National Marine Sanctuary (FKNMS) were conducted over several years and provide the basis for computing the intrinsic recovery rate and thus, the replacement ratio. This computation is performed using the Habitat Equivalency Analysis (HEA) and determines the lost on-site services pertaining to the ecological function of an area as the result of an injury and sets this against the difference between intrinsic recovery and recovery afforded by restoration. Joining empirical field data with economic theory has produced a reasonable and typically conservative means of determining the level of restoration and this has been fully supported in Federal Court rulings. Having clearly defined project goals allows application of the success criteria in a predictable, consistent, reasonable, and fair manner.
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Genetic variation, its assessment and implications to the conservation of seagrasses
Article
  • Feb 2002
  • MOL ECOL
In a study of the widespread Australian endemic seagrass Posidonia australis, allozyme analysis identified a wide range in population genetic structure assessed using the multilocus genotype diversity statistic (DG). Values of DG between zero and one were obtained; however, RAPD analysis generally detected higher levels of diversity, where DG values were all greater than 0.5 (DG = 0.67 – 1). Some populations were allozymically monomorphic using allozyme analysis yet were highly polymorphic using RAPD analysis. The differences observed between methods, particularly among allozymically uniform populations, demonstrate the importance of choosing an appropriate method when assessing genotypic diversity. Different methods may reflect different historical aspects of population processes where allozymes reflect broader-scale gene flow and population establishment and DNA fingerprinting methods such as RAPDs may reflect fine-scale local recruitment events and shorter-term population processes. Using either method alone, particularly in genotypically depauperate organisms such as seagrasses and other clonal organisms, will be problematic in assessing their population genetic potential, a parameter being used by conservation managers to decide upon management strategies in rare and endangered organisms. It is recommended that the impact of disturbance assessed using genotypic diversity measures requires more than one technique to provide the most appropriate information for designing subsequent conservation strategies.
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