A study of the variable species Oxyrrhynchium hians s.l. in NW Europe based on nuclear ITS, and plastid rpl16 and trnL‑trnF, as well as morphology, revealed unsuspected species level diversity. Three taxa are distinguishable by morphology: O. distichum with complanate or sub-complanate branch leaves and long and narrow leaf lamina cells, O. hians with cordate or broadly ovate, concave leaves that are evenly arranged around the stems and branches, and O. swartzii with mostly complanate or sub-complanate branch leaves and compared with O. distichum relatively short and wide leaf lamina cells. In Sweden O. distichum grows almost exclusively on base-rich or calcareous rocks and has been recorded from a belt stretching from the Baltic Sea islands of Öland and Gotland to Dalarna and southernmost Norway, whereas the other two species grow on various substrates and have wider distributions. Oxyrrhynchium hians grows in more nutrient-rich habitats than O. swartzii and is therefore absent from regions with relatively poor soils. Oxyrrhynchium swartzii occurs northwards to Sør-Trøndelag in Norway and Jämtland and Medelpad in Sweden and includes two semi-cryptic species that differ slightly in size and may have relatively more western and eastern distributions, respectively, in Fennoscandia.

For the molecular portion of this study, 32 specimens of O. hians s.l. from Sweden, six from Norway, and three from Switzerland were included. One specimen each of the two closely related species O. schleicheri (R. Hedw.) Röll and O. speciosum (Brid.) Warnst., two other unambiguous members of Oxyrrhynchium (Ignatov and Huttunen 2002, Ignatov and Isoviita 2003), were included as outgroup.

Total DNA was extracted using the NucleoMag® Plant kit for DNA isolation from plant tissue (Macherey-Nagel) with the KingFisher Duo magnetic particle processor. Double stranded DNA templates were prepared by polymerase chain reaction (PCR). PCR was performed using IllustraTM Hot Start Mix RTG (GE Healthcare) in a 25 µl reaction volume according to the manufacturer’s instructions.

In all cases, the specified PCR programs were initiated by a denaturation step of 5 min at 95º C and followed by a final extension period of 10 min at 72º C. The PCR programs were, for the nuclear internal transcribed spacers 1 and 2 (ITS), 4 cycles of 30 sec at 95º C, 40 sec at 55º C, and 1 min at 72º C, 4 cycles of 30 sec at 95º C, 30 sec at 53º C, and 1 min at 72º C, 35 cycles of 30 sec at 95º C, 30 sec at 50º C, and 1 min at 72º C, with the primers ‘ITS4bryo’ (Stech 1999) and ‘ITSbryoR’ (Hedenäs 2014). For the plastid rpl16 G2 intron (rpl16), 43 cycles of 30 sec at 95º C, 30 sec at 58º C, and 1 min 15 sec at 72º C, with the primers ‘F71’ (Jordan, et al. 1996) and ‘rpl16-antR2’ (Hedenäs 2008), and for the plastid trnLUAA intron plus trnLUAA-trnFGAA spacer (trnL-trnF), 4 cycles of 30 sec at 95º C, 40 sec at 57º C, and 1 min at 72º C, 4 cycles of 30 sec at 95º C, 30 sec at 55º C, and 1 min at 72º C, 35 cycles of 30 sec at 95º C, 30 sec at 52º C, and 1 min at 72º C, with the primers ‘trnC’ and ‘trnF’ (Taberlet, et al. 1991).

The amplified PCR products were purified from excess primers and nucleotides by adding 1µl of Exonuclease I (20U/µl) and 4µl of FastAP Thermosensitive Alkaline Phosphatase (1U/µl) (Thermo Scientific™) and incubating at 37° C for 30 min followed by an enzyme inactivation step at 80° C for 15 min. The purified PCR products, together with the same primers used for PCR amplification, were subsequently sent to Macrogen Europe B.V (Amsterdam, The Netherlands) for single-stranded sequencing on an Applied Biosystems 3730XL sequencer.

Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE® 0.9971 (http://www.phyde.de/index.html; accessed 17 February 2023). The assembled sequences were aligned manually in PhyDE®. Regions of partially incomplete data in the beginning and end of the sequences were identified and were excluded from subsequent analyses. Gaps were coded using the simple indel coding of Simmons and Ochoterena (2000) in SeqState (Müller 2005).