Earlier studies on Isothecium s.l. suggested potential interchange of genetic material between species that are nowadays known to belong to separate genera. In this study, we analyze the molecular variation in three molecular markers (nuclear gdp; mitochondrial nad5; and plastid trnG) in a wide selection of Isothecium alopecuroides and Pseudisothecium myosuroides specimens to infer whether this exchange of genetic material is frequent. In addition, we explore the phylogeographic structure of these two species in Europe. Our results show repeated genetic interchange for the nuclear and mitochondrial markers in both species, with intergeneric hybridization as the most probable explanation. The genetic lineages found indicate a complex biogeographical history for I. alopecuroides, including both postglacial immigration from different glacial refugia and developing haplotypes potentially adapted to cold climates. As for P. myosuroides, genetic variants could either indicate a postglacial immigration history or adaptation to oceanic conditions. Further studies including a larger set of more variable molecular markers could help to reach final conclusions on the results here presented.

We included 94 Isothecium alopecuroides and 40 Pseudisothecium myosuroides specimens, and 13 additional members of the Lembophyllaceae lineage to which these two species belong: Isotheciastrum subdiversiforme (1), Isothecium algarvicum (2), Pseudisothecium cristatum (1), P. holtii (3), P. interludens (3), P. prolixum (2), and P. stoloniferum (1). Most sequences were newly generated for the present study, and some came from an earlier study. We studied a portion of the nuclear glyceraldehyde 3-phosphate dehydrogenase (gdp), the mitochondrial nad5 G1 intron (nad5), and the plastid trnG_UCC G2 intron (trnG).

Total DNA was extracted using the DNeasy® Plant Mini Kit for DNA isolation from plant tissue (Qiagen) according to the manufacturer’s instructions. PCR was performed using Ready-To-Go^TM PCR Beads (Amersham Pharmacia Biotech) in a 25 ml reaction volume, also following the manufacturer’s protocol. PCR protocol for nad5: 1x 95º 5 min; 1x TC 94º 40 sec, 60‑54º 40 sec, 72º 1 min 30 sec; 34x 94º 30 sec, 54º 30 sec, 72º 1 min 30 sec; 1x 72º 10 min, with the primers nad5-F4, nad5-R3 (Cox et al. 2004), nad5-Bryo-F160 (ATCCTCAGAGACTGTACGTG), nad5-Bryo-R645 (GATGCTGCGTCCCTAACAACTA). PCR protocol for gdp: 1x 95º 5 min; 1x TC 94º 40 sec, 60‑54º 40 sec, 72º 1 min 30 sec; 34x 94º 30 sec, 54º 30 sec, 72º 1 min 30 sec; 1x 72º 10 min, with the primers: gdp1790F, gdp3050Rr (Wall 2002), or 1x 95º 5 min; 1x TC 94º 40 sec, 60‑54º 40 sec, 72º 1 min 30 sec; 40x 94º 30 sec, 54º 30 sec, 72º 1 min 30 sec; 1x 72º 10 min, with the primers gdp-Iso-254F (GCTCGCAAAGGTATCATATC), gdp-Iso-784R (CACCTTGCCCACAGCCTTG). PCR protocol for trnG: 1x 95º 5 min; 38x 95º 30 sec, 52º 40 sec, 72º 1 min 30 sec; 1x 72º 8 min, with the primers trnGf, trnGr (Werner et al. 2009).

Amplified fragments were purified using one portion of Exonuclease I (EXO I) (20 u ml^-1) plus four portions of FastAP^TM Thermosensitive Alkaline Phosphatase (1 u ml^-1) (Fermenta LIFE SCIENCE). For 20 ml PCR product, 1 ml EXO I + 4 ml FastAP^TM were used. Sequencing was performed using the ABI BigDye Terminator Kit (Applied Biosystems) according to the manufacturer’ instructions (BDT ver. 3.1). Sequencing products were cleaned using the DyeEx^® 96 Kit (QUIAGEN) with the same primers used for the initial PCRs. Sequencing products were resolved on an ABI3130xl automated sequencer. Double-stranded sequencing was performed in all cases.

Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE® 0.9971 (http://www.phyde.de/index.html; accessed 22 November 2018). The assembled sequences were aligned manually in PhyDE®. Regions of partially incomplete data at 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).