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Identifying sex in non-fertile individuals of the moss Drepanocladus turgescens (Bryophyta: Amblystegiaceae) using a novel molecular approach

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Abstract

Sex identification before sexual maturity is notoriously difficult in plants with separate sexes, but is crucial to address many life history related issues. To study the performance of the two sexes in the rarely sexually reproducing dioecious moss Drepanocladus turgescens a molecular sex marker is needed. The female-targeting marker previously developed for D. trifarius and D. lycopodioides amplifies for a few D. turgescens males, which can thus not be distinguished from females. In a significant addition to the earlier developed method we sequenced the portion successfully amplified by the primers PT−3f and PT−3r for six females and three males. Differences between males and females were revealed at five sequence positions. Examination of a total of fourteen females and seven marker amplifying males confirm that females and such males differ consistently at these positions. The usefulness of a previous protocol for moss sex identification is thus extended to another dioecious moss by the addition of a step where a portion of the sex-correlated region is sequenced.

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Fig. 1

References

  • Barrett SCH, Hough J (2013) Sexual dimorphism in flowering plants. J Exp Bot 64:67–82

    Article  CAS  PubMed  Google Scholar 

  • Bertiller MB, Sain CL, Bisigato AJ, Coronato FR, Ares JO, Graff P (2002) Spatial sex segregation in the dioecious grass Poa ligularis in northern Patagonia: the role of environmental patchiness. Biodivers Conserv 11:69–84

    Article  Google Scholar 

  • Bisang I, Hedenäs L (2005) Sex ratio patterns in dioicous bryophytes re-visited. J Bryol 27:207–219

    Article  Google Scholar 

  • Bisang I, Hedenäs L (2013) Males are not shy in the wetland moss Drepanocladus lycopodioides. Int J Plant Sci 174:733–739

    Article  CAS  Google Scholar 

  • Bisang I, Korpelainen H, Hedenäs L (2010) Can the sex-specific molecular marker of Drepanocladus trifarius uncover gender in related species? J Bryol 32:305–308

    Article  Google Scholar 

  • Bisang I, Ehrlén J, Persson C, Hedenäs L (2014) Family affiliation, sex ratio and sporophyte frequency in unisexual mosses. Bot J Linn Soc 174:163–172

    Article  Google Scholar 

  • Bisang I, Ehrlén J, Korpelainen H, Hedenäs L (2015) No evidence of sexual niche partitioning in a dioecious moss with rare sexual reproduction. Ann Bot 116:771–779. doi:10.1093/aob/mcv133

    Article  PubMed  PubMed Central  Google Scholar 

  • Brzyski JR, Taylor W, McLetchie DN (2014) Reproductive allocation between the sexes, across natural and novel habitats, and its impact on genetic diversity. Evol Ecol 28:247–261

    Article  Google Scholar 

  • Cronberg N (2002) Colonization dynamics of the clonal moss Hylocomium splendens on islands in a Baltic land uplift area: reproduction, genet distribution and genetic variation. J Ecol 90:925–935

    Article  Google Scholar 

  • During HJ (1992) Ecological classification of bryophytes and lichens. In: Bates JW, Farmer AM (eds) Bryophytes and lichens in a changing environment. Clarendon Press, Oxford, pp 1–31

    Google Scholar 

  • Hedenäs L, Bisang I (2011) The overlooked dwarf males in mosses: unique among green land plants. PPEES 13:121–135

    Google Scholar 

  • Hedenäs L, Bisang I (2013) Do male and female plants display different haplotype patterns in the moss Drepanocladus trifarius (Bryophyta, Amblystegiaceae)? Pol Bot J 58:279–285

    Google Scholar 

  • Hedenäs L, Bisang I, Korpelainen H, Cronholm B (2010) The true sex ratio in European Pseudocalliergon trifarium (Bryophyta: Amblystegiaceae) revealed by a novel molecular approach. Biol J Linn Soc 100:132–140

    Article  Google Scholar 

  • Korpelainen H, Bisang I, Hedenäs L, Kolehmainen J (2008) The first sex-specific molecular marker discovered in the moss Pseudocalliergon trifarium. J Hered 99:581–587

    Article  CAS  PubMed  Google Scholar 

  • McDaniel SF, Perroud P-F (2012) Invited perspective: bryophytes as models for understanding the evolution of sexual systems. Bryologist 115:1–11

    Article  Google Scholar 

  • McDaniel SF, Willis HJ, Shaw AJ (2007) A linkage map reveals a complex basis for segregation distortion in an interpopulation cross in the moss Ceratodon purpureus. Genetics 176:2489–2500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McLetchie DN (2001) Sex-specific germination response in the liverwort Sphaerocarpos texanus (Sphaerocarpaceae). Bryologist 104:69–71

    Article  Google Scholar 

  • Newton ME (1971) A cytological distinction between male and female Mnium undulatum Hedw. Trans Brit Bryol Soc 6:230–243

    Article  Google Scholar 

  • Norell TE, Jones KS, Payton AC, McDaniel SF (2014) Meiotic sex ratio variation in natural populations of Ceratodon purpureus (Ditrichaceae). Am J Bot 101:1572–1576

    Article  Google Scholar 

  • Obbard DJ, Harris SA, Pannell JR (2006) Sexual systems and population genetic structure in an annual plant: testing the metapopulation model. Am Nat 167:354–366. doi:10.1086/499546

    Article  PubMed  Google Scholar 

  • Shaw AJ, Beer SC (1999) Life history variation in gametophyte populations of the moss Ceratodon purpureus. Am J Bot 86:512–521

    Article  CAS  PubMed  Google Scholar 

  • Stark LR, McLetchie DN, Eppley SM (2010) Sex ratios and the shy male hypothesis in the moss Bryum argenteum (Bryaceae). Bryologist 113:788–797

    Article  Google Scholar 

  • Stehlik I, Friedman J, Barrett SCH (2008) Environmental influence on primary sex ratio in a dioecious plant. Proc Natl Acad Sci USA 105:10847–10852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank K. Mirbakhsh and M. Pietiläinen for their efficient labwork. Two reviewers provided valuable comments to an earlier version of the manuscript.

Author information

Authors and Affiliations

  1. Department of Botany, Swedish Museum of Natural History, Box 50007, 104 05, Stockholm, Sweden

    Lars Hedenäs & Irene Bisang

  2. Department of Agricultural Sciences, University of Helsinki, PO Box 27, 00014, Helsinki, Finland

    Helena Korpelainen

Authors
  1. Lars Hedenäs
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  2. Helena Korpelainen
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  3. Irene Bisang
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Corresponding author

Correspondence to Lars Hedenäs.

Additional information

Membership of the Botanical Society of Japan: One of the authors, Helena Korpelainen, is a member.

Appendices

Appendix 1: Protocol for gender identification in the moss Drepanocladus turgescens

For each shoot to be sexed, extract total DNA using the DNeasy® Plant Mini Kit (Qiagen). Amplify the markers using Ready-To-GoTM PCR Beads (Amersham Pharmacia Biotech).

Sex identification, Step 1: Run a PCR with the primers PT−3f (5#-GGA TTG ATA TTG GCA TTG AGT T-3#) and PT−3r (5#-TGG AAT GTC ACA TTG TTT AGG A-3#) (Korpelainen et al. 2008). Use 2 µL of template and the PCR protocol 4 min at 94 °C for initial denaturation, followed by 35 cycles of 45 s at 94 °C, 45 s at 53 °C, and 30 s at 72 °C, ending with a final extension step of 8 min at 72 °C. For samples that do not amplify, check DNA quality by running a second PCR with the primers PT−1f (5#-TTC CTA GTG GGG AAC AGA AAA A-3#) and PT−2r (5#-TAAGCGGAAAAA TGGGAT TAGA-3#) (Korpelainen et al. 2008). Use the same template amount and PCR protocol as for PT−3f and PT−3r.

Specimens that did not amplify in the first PCR, and yielded PCR products in the second are male. The other specimens could be either male or female.

Sex identification, Step 2: Sequence the region amplified by the PT−3f and PT−3r primers. Clean the amplified fragments with a DyeEx® 96 Kit (QIAGEN) using 1 portion Exonuclease I (EXO I) 20 µ/µl + 4 portions FastAP TM Thermosensitive Alkaline Phosphatase 1 µ/µl (Fermenta LIFE SCIENCE). For 20 µl PCR reaction, use 1 µl EXO I + 4 µl FastAP TM. Cycle sequencing is done with the ABI BigDye Terminator Kit (Applied Biosystems) according to the instructions on the kit (BDT ver. 2.0 or 3.1), and products are cleaned using the DyeEx 96 Kit (QIAGEN). Resolve the sequencing products (we used an ABI 3100 automated sequencer).

Edit and assemble nucleotide sequence fragments for each DNA region and align the sequences using suitable software (we used PhyDE® 0.9971; http://www.phyde.de/index.html). Check the bases at positions 11 (male = gap, female = C), 34 (G, A), 51 (G, A), 55 (T, C), and 57 (G, A), position 1 being the first one after the primer PT−3f, to identify the sex of the respective specimens (see Fig. 1).

Appendix 2

Voucher specimens, sex, amplification or not with PT−3f and PT−3r, locality (lat., long.), collector (number), herbarium: (herbarium reg. no.). When amplification occurred and sequences were successfully retrieved, the latter are found in Fig. 1. Abbreviations: m = male; f = female; PT+/PT− = amplifying or not with PT−3f and PT−3r. Samples PT1001–PT1007 were used initially, these and samples PT1007b, PT1010, PT1026, PT1027, PT1031, PT1032, PT1035, and PT1036 were used to characterize male and female sequences for the region amplified by PT−3f and PT−3r, and the remaining 34 specimens were used to verify the sex-related differences among the sequences (see “Materials and methods”).

PT1001: m, PT-, Sweden. Öland, Mörbylånga (+56.6545, +16.5712), K.Hylander 254, K.H, private herb.; PT1002: m, PT−, Sweden. Öland, SE of Eriksöre (+56.6112, +16.4958), L. Hedenäs, S: B174624; PT1003: m, PT+, Sweden. Gotland, Bunge (+57.8791, +19.0071), L. Hedenäs, S: B1124; PT1004: f, PT+, Sweden. Öland, Resmo (+56.5534, +16.4543), L. Hedenäs, S: B174629; PT1005: f, PT+, Sweden. Öland, Stenåsa (+56.5199, +16.5510), L. Hedenäs, S: B174790; PT1006: f, PT+, Sweden. Torne Lappmark, Torneträsk area (+68.3243, +18.8360), L. Hedenäs, S: B39204; PT1007: m, PT+, Sweden. Gotland, Bunge (+57.8791, +19.0071), L. Hedenäs, S: B1124; PT1007b: f, PT+, Sweden. Gotland, Othem (+57.7290, +18.6871), L. Hedenäs, S: B184192; PT1008: f, PT+, Sweden. Öland, Räpplinge (+56.8189, +16.6158), L. Hedenäs & I. Bisang, S: B184235; PT1010: m, PT-, Sweden. Öland, Räpplinge (+56.8150, +16.6057), L. Hedenäs & I. Bisang, S: B184239; PT1012: f, PT+, Sweden. Öland, Räpplinge (+56.8150, +16.6057), L. Hedenäs & I. Bisang, S: B184238; PT1014: f, PT+, Sweden. Öland, Torslunda (+56.6132, +16.4948), L. Hedenäs & I. Bisang, S: B184243; PT1015: m, PT-, Sweden. Öland, Resmo (+56.5564, +16.4660), L. Hedenäs & I. Bisang, S: B184241; PT1019: m, PT-, Sweden. Öland, Stenåsa (+56.5272, +16.5249), L. Hedenäs & I. Bisang, S: B184246; PT1022: f, PT+, Sweden. Öland, Vickleby (+56.5608, +16.4707), L. Hedenäs & I. Bisang, S: B184240; PT1023: f, PT+, Sweden. Gotland, Fleringe (+57.8880, +18.9744), L. Hedenäs, S: B184254; PT1024: m, PT+, Sweden. Gotland, Fleringe (+57.8880, +18.9744), L. Hedenäs, S: B184254; PT1025: f, PT+, Sweden. Gotland, Fleringe (+57.8892, +18.9777), L. Hedenäs, S: B184253; PT1026: m, PT+, Sweden. Gotland, Fleringe (+57.8892, +18.9777), L. Hedenäs, S: B184253; PT1027: f, PT+, Sweden. Jämtland, Aspås (+63.4403, +14.5103), L. Hedenäs, S: B184255; PT1028: m, PT+, Sweden. Gotland, Bunge (+57.8791, +19.0070), L. Hedenäs, S: B184252; PT1029: f, PT+, Sweden. Gotland, Bunge (+57.8791, +19.0070), L. Hedenäs, S: B184252; PT1031: m, PT-, Sweden. Gotland, Rute (+57.8422, +18.9274), L. Hedenäs, S: B184251; PT1032: f, PT+, Sweden. Gotland, Rute (+57.8422, +18.9274), L. Hedenäs, S: B184251; PT1033: f, PT+, Sweden. Gotland, Othem (+57.7596, +18.7028), L. Hedenäs, S: B184249; PT1035: f, PT+, Estonia. Saarema, Kingissepa (+58.1031, +22.1875), L. Hedenäs, S: B39453; PT1036: m, PT−, Estonia. Saarema, Kingissepa (+58.1031, +22.1875), L. Hedenäs, S: B39454; PT1037: m, PT−, Sweden. Härjedalen, Storsjö (+62.8896, +12.6916), N. Hakelier, S: B39106; PT1038: m, PT+, Canada. Quebec, Mingan Archipelago Nat. Park Reserve (+50.2490, −64.0062), T.A.Hedderson 8383, S: B193810; PT1039: m, PT−, Canada. Alberta. Mountain Park Area (ca. +52.91, −117.38), D. H. Vitt & W. Peterson 5498, S: B193811; PT1040: f, PT+, United States. Minnesota, Becker County (+47.1153, −95.9356), L. Hedenäs & J. A. Janssens, S: B16959; PT1041: m, PT−, United States. Alaska. Alaska Range District (ca. +63.44, −150.86), F. J. Hermann 21381, S: B194005; PT1042: f, PT+, Canada. Newfoundland, Northern Peninsula (ca. +51.62, −55.90), T. Hedderson 505 (Bryoph. Exs. T.-N. et Labr. 15), S: B194006; PT1043: f, PT+, Canada. Yukon Territory, Lake Alsek (ca. +60.75, −137.51), H. Crum & W. B. Schofield 8566, S: B194007, PT1045: m, PT+, Sweden. Gotland, Lärbro (+57.78, +18.86), L. Hedenäs, S: B196928; PT1046: m, PT−, Sweden. Gotland, Fleringe (+57.87, +18.92), L. Hedenäs & I. Bisang, S: B196905; PT1047: m, PT−, Sweden. Pite Lappmark, Arjeplog (+66.88, +16.17), G. Een, S: B79200; PT1048: m, PT−, Greenland. Scoresby Sund (ca. +70.25, −29.00), K. Holmen 18615, S: B200739; PT1049: m, PT−, Sweden. Gotland, Ardre (+57.36, +18.72), L. Hedenäs & I. Bisang, S: B204947; PT1300: m, PT−, Norway. Nord-Trøndelag, Røyrvik (+64.88, +13.81), L. Hedenäs, S: B205273; PT1301: m, PT−, USA. New York, Jefferson Co., Wartertown (+44.09, −76.11), I. Bisang 86745, S: B205489; PT1302: m, PT+, Sweden. Härjedalen, Tännäs (+62.74, +12.46), L. Hedenäs, S: B208650; PT1303: m, PT−, Sweden. Härjedalen, Tännäs (+62.75, +12.45), L. Hedenäs, S: B208654; PT1304: m, PT-, Sweden. Härjedalen, Tännäs (+62.73, +12.45), L. Hedenäs, S: B208661; PT1305: m, PT−, Sweden. Öland, Mörbylånga (+56.50, +16.45), L. Hedenäs, S: B210724; PT1306: m, PT−, Sweden. Öland, Kastlösa (+56.48, +16.46), L. Hedenäs, S: B210735; PT1307: m, PT−, Sweden. Öland, Kastlösa (+56.48, +16.47), L. Hedenäs, S: B210736; PT1308: m, PT−, Sweden. Öland, Kastlösa (+56.48, +16.48), L. Hedenäs, S: B210737; PT1309: m, PT−, Sweden. Öland, Kastlösa (+56.47, +16.49), L.Hedenäs, S: B210744.

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Hedenäs, L., Korpelainen, H. & Bisang, I. Identifying sex in non-fertile individuals of the moss Drepanocladus turgescens (Bryophyta: Amblystegiaceae) using a novel molecular approach. J Plant Res 129, 1005–1010 (2016). https://doi.org/10.1007/s10265-016-0837-9

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