11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.14.97299729Data PaperAscomycotaLecanoraceaeLecanoralesLecanoromycetesPopulation geneticsWorldCharacterization of microsatellite markers in the cosmopolitan lichen-forming fungus Rhizoplacamelanophthalma (Lecanoraceae)LindgrenHanna1hlindgren@fieldmuseum.orgLeavittSteven D.1https://orcid.org/0000-0002-5034-9724LumbschH. Thorsten1Integrative Research Center, The Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605, USAThe Field Museum of Natural HistoryChicagoUnited States of America
Corresponding author: Hanna Lindgren (hlindgren@fieldmuseum.org)
Academic editor: P. Divakar
201631082016143136FFB98421-EE50-FFC3-0600-FFB4FFF8AE0F1486953006201623082016Hanna Lindgren, Steven D. Leavitt, Thorsten LumbschThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Rhizoplacamelanophthalma s.l. is a group of morphologically distinct and chemically diverse species that commonly occur in desert, steppe and montane habitats worldwide. In this study, we developed microsatellite markers to facilitate studies of genetic diversity, population structure, and gene flow in the nominal taxon of this group, Rhizoplacamelanophthalma. We characterized 10 microsatellite markers using a draft genome of R.melanophthalma s. str. assembled from Illumina reads. These loci were tested for 21 R.melanophthalma s. str. specimens and also with a subset of 18 specimens representing six additional species in the R.melanophthalma complex. The number of alleles per locus in R.melanophthalma s. str. ranged from 3 to 11 with an average of 6.7. Nei’s unbiased gene diversity ranged from 0.35 to 0.91. Amplifications of the microsatellite loci were largely successful in the other six species, although only three markers were found to be polymorphic. The new markers will provide an additional resource for studying genetic, population- and landscape-level processes in the cosmopolitan taxon Rhizoplacamelanophthalma s. str.
Lindgren H, Leavitt SD, Lumbsch HT (2016) Characterization of microsatellite markers in the cosmopolitan lichen-forming fungus Rhizoplaca melanophthalma (Lecanoraceae). MycoKeys 14: 31–36. doi: 10.3897/mycokeys.14.9729
Introduction
Rhizoplacamelanophthalma (DC.) Leuckert & Poelt s.l. represents a group of morphologically distinct and chemically diverse species of lichen-forming fungi with broad ecological and geographical distributions. Species in this group occur all over the world in disjunct populations in continental climates, although species in this complex are notably absent from Australia. Rhizoplacamelanophthalma s.l. commonly grows on siliceous or calcareous rock in arid climates, but can also be found in montane coniferous forests, alpine tundra habitats, and bi-polar populations in the Arctic and Antarctica (McCune 1987). Members of this group are commonly used in air-quality biomonitoring research, making it an important species for conservation (Aslan et al. 2004; Dillman 1996). The species complex belongs to the recently re-circumscribed monophyletic genus Rhizoplaca in Lecanoraceae (Zhao et al. 2016).
Previous multi-locus and phylogenomic studies support the circumscription of multiple species within R.melanophthalma s.l. (Leavitt et al. 2011, 2013, 2016b), many of which occur in sympatry in Western North America. In Western North America the distribution area of these species extends from the northern boreal zone to Mexico along the Rocky Mountains with a center of diversity in the Great Basin region (Leavitt et al. 2011). Rhizoplacamelanophthalma s. str. has the broadest ecological and geographic distribution of all known species within this complex, with populations occurring in desert, montane and steppe ecosystems in Antarctica, Central Asia, Europe, and North and South America (Leavitt et al. 2013).
The Rhizoplacamelanophthalma group provides an interesting system for assessing genetic diversity, population structure and gene flow in symbiotic fungal species with broad ecological and geographic distributions. To facilitate additional research into population- and landscape-level processes, 10 microsatellite markers were developed for R.melanophthalma s.str.
Materials and methods
A total of 42 specimens representing seven different species in the Rhizoplacamelanophthalma species complex were included in this study. Twenty-one of these represented R.melanophthalma s. str., three R.haydenii, four R.novomexicana, two R.parilis, four R.polymorpha, six R.porteri and two R.shushanii (Table 1). DNA was extracted from these specimens as described previously (Leavitt et al. 2011).
Voucher information for Rhizoplaca specimens used in this study. Herbaria codes are provided for each specimen in parentheses following voucher number.
Species
DNA No.
Voucher
Locality
R.melanophthalma
8639c
Leavitt 2013-CO-CP-8639C (F)
USA, CO
R.melanophthalma
8639d
Leavitt 2013-CO-CP-8639D (F)
USA, CO
R.melanophthalma
8654a
Leavitt 2013-CO-RM-8654A (F)
USA, CO
R.melanophthalma
8654b
Leavitt 2013-CO-RM-8654B (F)
USA, CO
R.melanophthalma
8663B
Leavitt 8663 (F)
USA, UT
R.melanophthalma
8663j
Leavitt-8663 (F)
USA, UT
R.melanophthalma
8665b
Leavitt-8665 (F)
USA, NV
R.melanophthalma
8665e
Leavitt-8665 (F)
USA, NV
R.melanophthalma
8665i
Leavitt-8665 (F)
USA, NV
R.melanophthalma
8665M
Leavitt-8665 (F)
USA, NV
R.melanophthalma
8668b
Leavitt-8668 (F)
USA, NV
R.melanophthalma
8668f
Leavitt-8668 (F)
USA, NV
R.melanophthalma
8668q
Leavitt-8668 (F)
USA, NV
R.melanophthalma
8668s
Leavitt-8668 (F)
USA, NV
R.melanophthalma
8668w
Leavitt-8668 (F)
USA, NV
R.melanophthalma
6026
H9203303 (F)
Kyrgyzstan, Ala-Buka
R.melanophthalma
6029
H9203135 (F)
Kyrgyzstan, Panfilov District
R.melanophthalma
6030
H9203327 (F)
Kyrgyzstan, Chatkal
R.melanophthalma
6435
Vondrak 9409 (PRA)
Russia, Chelyabinsk
R.melanophthalma
6604
MAF-Lich 16805 (MAF)
Spain, Teruel
R.melanophthalma
6605
MAF-Lich 16778 (MAF)
Spain, Teruel
R.haydenii
8683
Leavitt 8683 (F)
USA, ID
R.haydenii
8935p
Leavitt 8935 (F)
USA, ID
R.haydenii
8935s
Leavitt 8935 (F)
USA, ID
R.novomexicana
8684a
Leavitt 8684A (F)
USA, NM
R.novomexicana
8684b
Leavitt 8684B (F)
USA, NM
R.novomexicana
8684c
Leavitt 8684C (F)
USA, NM
R.novomexicana
8684d
Leavitt 8684D (F)
USA, NM
R.parilis
8665N
Leavitt-8665 (F)
USA, NV
R.parilis
8665u
Leavitt-8665 (F)
USA, NV
R.polymorpha
8668g
Leavitt-8668 (F)
USA, NV
R.polymorpha
8668l
Leavitt-8668 (F)
USA, NV
R.polymorpha
8668p
Leavitt-8668 (F)
USA, NV
R.polymorpha
8668r
Leavitt-8668 (F)
USA, NV
R.aff.porteri
8665x
Leavitt-8665 (F)
USA, NV
R.aff.porteri
8668j
Leavitt-8668 (F)
USA, NV
R.aff.porteri
8668m
Leavitt-8668 (F)
USA, NV
R.porteri
8665t
Leavitt-8665 (F)
USA, NV
R.porteri
8668e
Leavitt-8668 (F)
USA, NV
R.porteri
8668h
Leavitt-8668 (F)
USA, NV
R.shushanii
8664A
Leavitt 13-TLM-001 (BRY-C)
USA, UT
R.shushanii
8664B
Leavitt 13-TLM-001 (BRY-C)
USA, UT
A draft genome of an axenic culture of R.melanophthalma was obtained from a previous study (Leavitt et al. 2016a). The program MSATCOMMANDER 1.0.8 (Faircloth 2008) was used to search for di-, tri-, tetra-, penta-, and hexanucleotide microsatellite repeats in contigs >5 kb from the draft assembly. Only repeats with a minimum length of 8 bp for dinucleotide repeats and 6 bp for the rest were accepted. A total of 244 scaffolds contained microsatellite repeats (87 di-, 127 tri-, 11 tetra-, 5 penta-, and 14 hexanucleotides). For 25 of these repeats, primers were designed with Primer3 (Rozen and Skaletsky 2000) as implemented in MSATCOMMANDER. An M13 tag (5’-TGTAAAACGACGGCCAGT-3’) was appended to forward primers and 5’ ends of the reverse primers were tailed with 5’-GTGTCTT-3’ tag.
Singleplex PCR reactions were performed in 10 µl reaction volumes consisting of 5.89 µl H2O, 1 µl 10x buffer (Roche Diagnostics, Indianapolis, USA), 0.6 µl 8 mM dNTP, 1 µl BSA, 0.15 µl Taq (Roche Diagnostics, Indianapolis, USA), 0.16 µl 6-FAM labeled M13 primer, 0.04 µl 10 µM M13 tailed forward primer, 0.16 µl 10 µM reverse primer, and 1 µl of genomic DNA. DNA amplification was performed using a touchdown PCR with initial denaturation at 95 °C for 5 min; followed by first 11 cycles of 30 s at 95 °C, 30 s at 60–50 °C, 1 min. at 72 °C, and then 35 cycles of 30 s at 95 °C, 30 s at 50 °C, 1.5 min. at 72 °C, and a final extension of 10 min. at 72 °C.
Fragment analysis was performed on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan-500 LIZ (Life Technologies, Warrington, UK) as an internal size standard. Genotyping was performed utilizing the microsatellite plugin in Geneious 9.1.2 (Biomatters Limited). Polymorphism within the microsatellites was tested in GenAlEx 6.5 (Peakall and Smouse 2012) by calculating Nei’s unbiased genetic diversity.
Results and discussion
Of the 25 microsatellites assessed, 18 amplified successfully and 10 were polymorphic in all 21 R.melanophthalma s. str. specimens (Table 2). The number of alleles per locus ranged from three to 11 with an average of 6.7. Nei’s unbiased genetic diversity varied between 0.353 and 0.919 with the average genetic diversity being 0.765 (Table 3). The same 18 microsatellites that amplified successfully with R.melanophthalma s. str. also amplified in R.haydenii, R.novomexicana, R.parilis, R.polymorpha, R.porteri, and R.shushanii, but only three loci were polymorphic in all these species. For these three loci (Rmel1, Rmel, 4, and Rmel8) the number of alleles ranged from 11 to 15 with the average of 13 and Nei’s unbiased genetic diversity varied between 0.892 and 0.905 with average of 0.900.
Microsatellite loci developed for Rhizoplacamelanophthalma s. str.
Locus
Primer sequences (5’–3’)
Repeat motif
Allele size range (bp)
GenBank accession no.
Rmel1
F: *GGCTGGGTGTTGTAGTGTTG R: ATACCTGGCGCTCAAGAATG
(GTT)18
179–203
KX755412
Rmel2
F: *TGGTGGATCTGAGAGCGTAC R: ACTTCAACCTTCACAACGCC
(CAGGCT)10
328–382
KX755413
Rmel3
F: *CAAAGGTCAGGAGAGGAGGG R: TGGACGCGTGGCAATTTATC
(AC)10
380–412
KX755414
Rmel4
F: *ATCGAGACTTACTTCCCGCC R: AATCGTATCTCCAGACCCGC
(GT)11
353–385
KX755415
Rmel5
F: *TTAGCCCGAGACCACATACG R: TGGAGAGATGAAGCTGGCTC
(CT)12
311–321
KX755416
Rmel6
F: *ACACCAGATCTCACTCAGGC R: CCGGGAGTAGGTGTAGATGC
(AC)10
184–192
KX755417
Rmel7
F: *TCCGGAACTGGCTTGATAGG R: CTGAAGTCGATGTTGGGAGC
(CCTT)11
314–362
KX755418
Rmel8
F: *TTTGCCCGACGTGCAATATC R: CTGCAGCACTCTAACCATGC
(AG)11
420–438
KX755419
Rmel9
F: *ATCTCCTGCATCTTCTCCGC R: AACGTCACATTCGCAGTCAC
(AC)10
309–331
KX755420
Rmel10
F: *TCATCACACCAGACACAGGG R: ACCTTAGGCCCAGACACATG
(AG)10
464–468
KX755421
M13 tail: TGTAAAACGACGGCCAGT.
Sample size, number of alleles (A) and Nei’s unbiased genetic diversity (He) of ten microsatellite loci developed for Rhizoplacamelanophthalma s. str.
Total
Locus
n
A
He
Rmel1
21
8
0.886
Rmel2
20
6
0.858
Rmel3
21
9
0.866
Rmel4
21
11
0.919
Rmel5
20
5
0.679
Rmel6
21
3
0.643
Rmel7
20
6
0.763
Rmel8
20
7
0.779
Rmel9
21
9
0.881
Rmel10
20
3
0.353
Average
6.7
0.765
The 10 polymorphic microsatellite markers for the lichen-forming fungus R.melanophthalma will help elucidate population processes that have led to the observed distribution patterns in this widespread species.
Acknowledgments
The authors would like to thank the Negaunee Foundation for providing funding for this study, and Kevin Feldheim (Pritzker Laboratory for Molecular Systematics) for assistance in the lab.
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