The Use of Dna Barcoding to Address Major Taxonomic Problems for Rare British Bryophytes

Home , Atrichum angustatum, Bryum knowltonii, Bryum warneum, Ceratodon purpureus, Ditrichum, Ditrichum cornubicum

THE USE OF DNA BARCODING TO ADDRESS MAJOR TAXONOMIC PROBLEMS FOR RARE BRITISH BRYOPHYTES

FINAL REVISED REPORT

FEBRUARY 2013

David Bell David Long Pete Hollingsworth

Royal Botanic Garden Edinburgh

With major contribution from D.T. Holyoak (Bryum)

CONTENTS

1. Executive summary……………………………………………………………… 3

2. Introduction……………………………………………………………………… 4

3. Methods 3.1 Sampling……………………………………………………………….. 6 3.2 DNA extraction & sequencing…………………………………………. 7 3.3 Data analysis…………………………………………………………… 9

4. Results 4.1 Sequencing success…………………………………………………….. 9 4.2 Species accounts 4.2.1 Atrichum angustatum ………………………………………… 10 4.2.2 Barbilophozia kunzeana ………………………………………13 4.2.3 Bryum spp……………………………………………………. 16 4.2.4 Cephaloziella spp…………………………………………….. 26 4.2.5 Ceratodon conicus …………………………………………… 29 4.2.6 Ditrichum cornubicum & D. plumbicola …………………….. 32 4.2.7 Ephemerum cohaerens ……………………………………….. 36 4.2.8 Eurhynchiastrum pulchellum ………………………………… 36 4.2.9 Leiocolea rutheana …………………………………………... 39 4.2.10 Marsupella profunda ……………………………………….. 42 4.2.11 Orthotrichum pallens & O. pumilum ……………………….. 45 4.2.12 Pallavicinia lyellii …………………………………………... 48 4.2.13 Rhytidiadelphus subpinnatus ……………………………….. 49 4.2.14 Riccia bifurca & R. canaliculata ………………………...... 51 4.2.15 Sphaerocarpos texanus ……………………………………... 54 4.2.16 Sphagnum balticum ………………………………………… 57 4.2.17 Thamnobryum angustifolium & T. cataractarum …………... 60 4.2.18 Tortula freibergii …………………………………………… 62

5. Conclusions……………………………………………………………………… 65

6. Dissemination of results………………………………………………………… 66

Acknowledgements………………………………………………………………… 66

References………………………………………………………………………….. 67

Appendix 1: Sample details and sequencing success (as attachment)

2 1. EXECUTIVE SUMMARY

This report assesses the taxonomic status and conservation value of 30 UK priority bryophyte species. These species were selected because they had some level of taxonomic uncertainty posing barriers to the implementation of conservation strategies. Molecular data from DNA barcoding markers were used to assess levels of genetic variation within priority species and between these species and close relatives. This data was then used in conjunction with morphological and ecological data to address the status of these species.

The key results are as follows: • The DNA barcoding data supports the effective species limits for 16 of the 30 priority species as recognised by the standard British bryophyte floras of Paton (1999) and Smith (2004). These species are Atrichum angustatum, Barbilophozia kunzeana, Bryum knowltonii, B. salinum, B. warneum, B. calophyllum, B. marratii, Ceratodon conicus, Ditrichum cornubicum, D. plumbicola, Marsupella profunda, Orthotrichum pallens, Rhytidiadelphus subpinnatus, Riccia canaliculata, Sphagnum balticum, T. freibergii . • The difficulty of identification of some these species suggests morphological characters currently employed are sometimes insufficient, but with intermediate or aberrant samples DNA barcode sequences can be used to resolve identification. • Bryum gemmiparum appears to contain two distinct lineages in the UK, with some populations apparently more closely related to the B. dichotomum complex than to the majority of B. gemmiparum populations. • The liverwort genus Cephaloziella appears to be very misunderstood and requires a full revision. • Despite the markedly different habitats occupied by populations of Eurhynchiastrum pulchellum and Pallavicinia lyellii in Britain, no genetic variation was found within either species to support the presence of cryptic species. • Leiocolea rutheana contains 2 distinct species and its var. laxa should be treated as a variety or synonym of L. gillmanii . • Orthotrichum pumilum was found to contain additional complexity with possible cryptic species which require further study. One lineage (including UK samples) within this O. pumilum complex appears to correspond to O. schimperi . • The available data suggest that Riccia bifurca may contain cryptic species with lineages in Britain differing genetically from the rest of Europe, but more data is required for a confident assessment. • No significant variation was found to support Thamnobryum angustifolium and T. cataractarum as distinct species, suggesting they are extreme habitat forms of T. alopecurum . • Sphaerocarpos texanus has been shown to contain 2 cryptic species, with separate lineages in Europe and North America. • Two species had to be excluded from the study. These were Cephaloziella baumgartneri (no suitable material available for sequencing) and Ephemerum cohaerens (few samples available and sequencing problems).

3 2. INTRODUCTION

Species based conservation programmes need to be based on a sound and stable taxonomic framework, both to avoid conservation action and resources being expended on dubious ‘species’ that turn out not to be distinct and also to avoid clearly distinct and endangered species not receiving conservation attention simply because their distinguishing characters are subtle. Both of these situations are likely to occur in bryophytes because of the shortage of expert bryological taxonomists, and the generally small and character-poor nature of bryophyte species.

Bryophytes (mosses, liverworts and hornworts) are the oldest land plants on earth and are very important ecologically. Britain has a rich bryophyte flora, containing approximately 69% of European bryophyte diversity. These small plants can be difficult to identify using morphological characters, with cryptic species being common, while their diminutive size often leads to them being overlooked by biologists, resulting in a general shortage of taxonomic expertise.

DNA barcoding involves using one or a few standard regions of DNA as a universal tool for species identification (Hebert et al. 2003). The ultimate goal is to develop a reference library of DNA barcode sequences against which any unknown samples can be compared for identification. The technique has a broad range of applications from general biological monitoring to identifying fragmented samples for forensic investigations and the illegal trade in endangered species, and it has also contributed to the discovery of new species. Previous research on British liverworts at RBGE has used DNA barcoding in bryophyte genera such as Anastrophyllum, Herbertus, Moerckia, Aneura and Conocephalum . These studies have demonstrated the power and efficiency of DNA barcoding for addressing these types of taxonomic issues, and for providing a robust framework for the interpretation of species morphologies, ecologies and distributions. This work has led to taxonomic clarification, a reappraisal of species limits and ranges, and the discovery of morphologically and ecologically distinct species which had previously been overlooked.

In the context of this study, taxonomic uncertainty associated with priority bryophyte species in the UK (species listed as being of principal importance in Section 41 of the NERC Act) has be characterised under six categories:

1) Species which are taxonomically problematical due to uncertainty over their distinctness from one (or a few) more common congeneric species (e.g. Riccia canaliculata ).

2) Species which are likely to consist of 2 or more distinct cryptic species (e.g. Eurhynchium pulchellum ).

3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus (e.g. Thamnobryum spp.).

4 4) Species which are problematic due to uncertainty over their distinctness and generic placement (hence requiring the study of species from multiple genera to solve the problem) (e.g. Bryum spp.).

5) The species is distinct, but its phylogenetic placement is uncertain.

6) There are no known/suspected taxonomic problems for a given species.

This project has focussed on species in categories 1-4, with the general aim of using a combination of DNA barcoding and examination of morphological characters to provide taxonomic clarification to underpin conservation programmes. A shortlist of 25 target species were selected whose taxonomic problems cause the greatest barriers to the implementation of conservation action, and which were considered to represent tractable problems within the timescale and resources of the project. The objectives of the project were as follows:

1) Coordinate a network of bryologists to collect DNA samples and voucher specimens of 25 taxonomically controversial priority bryophyte species and their relatives

2) Obtain preliminary identifications for all samples, and database sample information and voucher information in the Barcode of Life Datasystem (BOLD)

3) Obtain DNA barcode sequences from 2-4 DNA regions for all samples

4) Undertake analyses of the barcode data to provide a framework for establishing the taxonomic status and identity of all samples

5) Re-appraise the morphological, ecological and geographical characteristics of the different groups of samples in light of the DNA barcoding data

6) Revise the status of 25 taxonomically controversial priority bryophyte species

7) Disseminate the project findings to conservationists, amateur natural historians, members of the public interested in biodiversity, and the scientific community, via online species accounts & identification guides, a workshop, a presentation, a non- technical paper, a policy brief and scientific papers.

3. METHODS

The project followed the standard RBGE DNA barcoding protocol, with existing herbarium specimens supplemented with field work to obtain samples from multiple populations of the priority species, as well as other related taxa required to provide taxonomic clarification. DNA sequences (‘DNA Barcodes’) were obtained from 2-4 gene regions ( rbcL, matK, trnH-psbA , ITS2). The data was then analysed to identify clusters of related sequences and interpreted in light of associated morphological, ecological and distributional data to provide taxonomic clarification for each group.

5 3.1 SAMPLING

A list of 25 taxonomically controversial priority species was compiled initially, with five additional priority species added as a contingency in case of sampling or sequencing difficulties with any of the original species. Table 1 details the 30 target priority species and key related species. Additional related species were sometimes included where it was deemed they might enhance the analyses, particularly in the cases of notoriously difficult genera such as Bryum and Orthotrichum .

Table 1: Target UK priority species, taxonomic categories & key related species Problem species Taxonomic Key related species category Atrichum angustatum 1 Atrichum undulatum Barbilophozia kunzeana 1 Barbilophozia floerkei, B. barbata, B. quadriloba, Lophozia obtusa, L. sudetica, Tritomaria quinquedentata Bryum gemmiparum 3, 4 Bryum alpinum, B. dixonii, B. mildeanum, B. muehlenbeckii Bryum knowltonii 3, 4 Bryum algovicum var . rutheana, B. intermedium, B. riparium Bryum salinum 3, 4 Bryum archangelicum Bryum warneum 3, 4 Bryum arcticum, B. calophyllum, B. marratii Bryum calophyllum 3, 4 Bryum arcticum, B. warneum, B. marratii Bryum marratii 1 Bryum arcticum, B. warneum, B. calophyllum Ceratodon conicus 1 Ceratodon purpureus Ditrichum cornubicum 2, 3 Ditrichum lineare, D. plumbicola Ditrichum plumbicola 1, 3 Ditrichum lineare, D. cornubicum Ephemerum cohaerens 1, 3 Ephemerum recurvifolium, E. sessile, E. hibernicum, E. serratum, E. minutissimum Eurhynchiastrum pulchellum 2 Leiocolea rutheana 2 Leiocolea bantriensis, L. gillmanii Orthotrichum pallens 1, 3 O. pumilum, O. schimperi, O. stramineum, O. tenellum Orthotrichum pumilum 1, 3 O. pallens, O. schimperi, O. stramineum, O. tenellum Pallavicinia lyellii 2 Rhytidiadelphus subpinnatus 1 Rhytidiadelphus squarrosus, Loeskeobryum brevirostre Riccia bifurca 3 Riccia spp. Riccia canaliculata 1, 3 Riccia spp. Sphaerocarpos texanus 1, 2 Sphaerocarpos michelii Sphagnum balticum 1, 2, 3 Sphagnum fallax, S. tenellum Thamnobryum angustifolium 3 Thamnobryum spp. Thamnobryum cataractarum 3 Thamnobryum spp. Tortula freibergii 1, 2 Tortula cuneifolia, T. solmsii, T. marginata Marsupella profunda 3 Marsupella sprucei, M. boeckii, M. stableri, M. brevissima, M. alpina Cephaloziella baumgartneri 1, 3 Cephaloziella spp. Cephaloziella calyculata 1, 3 Cephaloziella spp. Cephaloziella integerrima 1, 3 Cephaloziella spp. Cephaloziella nicholsonii 1, 3 Cephaloziella spp. Taxonomic categories of priority species: 1) Species which are taxonomically problematical due to uncertainty over their distinctness from one (or a few) more common congeneric species. 2) Species which are likely to consist of 2 or more distinct cryptic species. 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus. 4) Species which are problematic due to uncertainty over their distinctness and generic placement (hence requiring the study of species from multiple genera to solve the problem).

6 Records of occurrence for each species throughout the UK were compiled from three sources: (a) the NBN (National Biodiversity Network), (b) the TBDB (Threatened Bryophytes Database) from JNCC through its co-ordinator Nick Hodgetts and (c) from herbarium specimens, primarily in E (herbarium of Royal Botanic Garden Edinburgh) and NMW (National Museum of Wales), Cardiff. Whenever possible herbarium specimens were used for DNA extraction, mainly from E and NMW, but loans of specimens were also provided by David T. Holyoak (personal herbarium), Tom L. Blockeel (personal herbarium), BM (herbarium of the Natural History Museum, London), MAUAM (Universidad Aut όnoma de Madrid) and LG (Université de Liège). Where no suitable herbarium material was available requests for freshly collected material were made to regional bryologists (see Appendix 1 for collectors used) or in some cases, where external collaborators were unavailable, DB and DGL went into the field to sample. Whenever possible, freshly collected material was dried in silica gel for optimum DNA preservation, with an associated air-dried herbarium voucher. Details of samples used can be found in Appendix 1.

3.2 DNA EXTRACTION & SEQUENCING

DNA was extracted and amplified using standard RBGE DNA barcoding protocols. Initially total DNA was extracted using Qiagen DNeasy Plant Mini Kits following the manufacturer’s protocol with an extended incubation at 65 °C. A Qiagen Qiaxtractor robot was later used for DNA extraction as this allowed the processing of larger batches of samples with increased efficiency. Four DNA barcoding regions were sequenced, including three plastid ( matK , rbcL and trnH-psbA ) and one nuclear (ITS2) marker. The combination of rbcL + matK forms the standard core barcode for land plants, established by the Consortium for the Barcode of Life (CBOL Plant Working Group 2009). trnH-psbA and ITS2 are alternative/supplementary barcoding regions proposed by Kress et al. (2005) and Chen et al. (2010) respectively. A combination of universal and lineage specific primers were used (Table 2). For rbcL the standard DNA barcode region was amplified using primers designed by Kress & Erickson (2007) and Fazekas et al. (2008). For trnH-psbA , as the spacer is very short in many bryophytes, instead of using the standard DNA barcoding primer sets designed by Tate & Simpson (2003) or Hamilton (1999), the trnH reverse primer designed by Sang et al. (1997) was used in combination with a forward primer located further back in the psbA gene (Forrest & Crandall-Stotler 2004). This was to maximise the amount of sequence data generated by each reaction. For ITS2, to reduce the risk of fungal contamination, primers designed from other bryophyte species were used (Stech et al. 2003; Olsson et al. 2009). There were no broadly applicable matK primer sets available for lower plants at the beginning of the project; however, another project based at RBGE, funded by the Gordon and Betty Moore Foundation, to develop and test primers for the matK barcode region in land-plants was being undertaken more or less concurrently and provided suitable primers for the liverworts and some moss groups before the end of the project.

7 Table 2: Details of primers used Region Primer Orientation Sequence Reference rbcL rbcL-aaf F 5’-ATGTCACCACAAACAGAGACTAAAGC-3’ Kress & Erickson (2007) rbcL ajf634r R 5'-GAAACGGTCTCTCCAACGCAT-3' Fazekas et al. (2008) matK matK-LivF1A F 5'-TYCATCCWGAAATTTTGATTCG-3' liverworts A. Forrest/P. Hollingsworth matK-LivR1A R 5'-ATAGTACTTTTRTGTTTACATGC-3' unpub. matK Moss404F F 5'-GGACTARYTATCAATCTATTCAYTC-3' A. Forrest/P. Hollingsworth mosses Moss1336R R 5'-TRCAAGCYAAYGTTTTAGC-3' unpub. trnH- psbA501F F 5’-TTTCTCAGACGGTATGCC-3’ Forrest & Crandall-Stotler psbA (2004) trnHR R 5’-GAACGACGGGAATTGAAC-3’ Sang et al. (1997) ITS SeqITS2 F 5'-AACAACTCTCAGCAACGG-3' Olsson et al. (2009) ITS4_bryo R 5'-TCCTCCGCTTAGTGATATGC-3' Stech et al. (2003)

Polymerase Chain Reaction (PCR) was performed in 10 µL reactions containing 1x PCR buffer and 0.75 U Taq polymerase (Bioline), 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 µM of each primer and 1 µL of template DNA. For rbcL and trnH-psbA Combinatorial Enhancer Solution (CES: 0.54 M betaine, 1.34% dimethyl sulfoxide [DMSO], 10 µg/µL bovine serum albumin [BSA]; Ralser et al. 2006) was added, and for ITS 4% DMSO was added to ameliorate the effects of secondary structure. For matK different protocols were adopted for moss and liverwort lineages. For mosses PCR reactions contained 1x PCR buffer, 0.2mM each dNTP, 1M betaine, 0.2M trehalose, 0.5µM each primer, 0.5U Phusion Taq (Finnzymes) and 1 µL of template DNA. For liverworts PCR reactions substituted Platinum Taq (Invitrogen) and added 2.5mM MgCl2. PCR conditions for each marker are given in Table 3. 2µL of ExoSAP-IT were added to 5µL of PCR product and run in a thermocycler at 37 ˚C for 30 mins followed by 15 mins at 80 ˚C for PCR clean-up. Sequences were run using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc.) on an ABI3730 automated sequencer at the GenePool Sanger Sequencing Service (University of Edinburgh, UK) using the same primers as for PCR. Sequencing PCR for ITS2 included 4% DMSO and for matK included 0.2M trehalose.

Table 3: PCR conditions Region Primer pair PCR conditions rbcL rbcL-aaf 4 min @ 94, 35x (60 sec @ 94, 60 sec @ 50, 90 sec @ 72), 10 min @ 72 rbcL ajf634r matK matK-Liv-F1A 4 min @ 94, 10x (30 sec @ 94, 30 sec @ 52, 60 sec @ 72), 25x (30 sec @ liverworts 88, 30 sec @ 48, 60 sec @ 72), 10 min @ 72 matK-Liv-R1A matK Moss404F 45 sec @ 98, 35x (10 sec @ 98, 30 sec @ 58, 40 sec @ 72), 10 min @ 72 mosses Moss1336R trnH- psbA501F 4 min @ 94, 2x (45 sec @ 94, 45 sec @ 50, 60 sec @ 72), 35x (45 sec @ psbA 94, 45 sec @ 45, 60 sec @ 72), 10 min @ 72 trnHR ITS SeqITS2 4 min @ 94, 30x (60 sec @ 94, 60 sec @ 55, 45 sec @ 72), 10 min @ 72 ITS4_bryo

8 3.3 DATA ANALYSIS

Bidirectional trace files were assembled and edited using Sequencher 4.7 (Gene Codes Corporation). Consensus sequences were aligned using ClustalW (Thompson et al. 1994) as implemented in MEGA 4 (Tamura et al. 2007). To visualise relationships among samples, maximum parsimony trees were produced in PAUP version 4.0b10 (Swofford 2003) for individual and combined datasets of rbcL , matK , trnH-psbA and ITS2. The maximum parsimony analysis consisted of an initial heuristic search using a TBR algorithm and 1000 random addition replicates, saving one tree per replicate; the most parsimonious trees were then used for a second heuristic search with TBR and a maximum of 1000 trees saved. Bootstrap analysis with 10,000 replicates was undertaken to assess levels of clade support.

4. RESULTS

4.1 SEQUENCING SUCCESS

In the course of the project 667 samples had DNA extracted and successfully sequenced for at least one DNA barcoding region, of a total of 714 samples attempted (93%). 60% of these were extracted from herbarium specimens and 40% from silica dried samples. The success rates for DNA extraction (successfully sequenced for at least one barcoding region) from silica dried material was 95%, compared to 86% from herbarium specimens. See appendix 1 for details of samples and sequencing success for each region.

rbcL and matK are the two ‘approved’ regions which form the core plant barcode (CBOL Plant Working Group 2009), while trnH-psbA and ITS2 were also included as supplementary barcoding regions for increased resolution. Sequencing of the rbcL barcode region was relatively straightforward, with 640 samples successfully sequenced (90% sequencing success rate). Species discrimination using rbcL alone was surprisingly high (ca. 80%) for bryophytes as compared to other groups of land plants, although in some genera, such as Bryum , resolution was very poor and additional barcoding regions are needed for species discrimination. Sequencing of the matK barcoding region has been problematic due to a lack of suitable bryophyte-specific primers to amplify the region and also the presence of a microsatellite in the centre of the matK barcode region in all moss genera except Sphagnum . A separate study at RBGE to develop lineage-specific matK primers for land plants was recently completed and separate matK primer sets were available for mosses and liverworts by the end of the project. For liverworts matK sequencing was generally straightforward using these primers but results for mosses were less consistent, with the supplied protocols and primers working well for some genera (e.g. Orthotrichum , Sphagnum ) but sequencing failing regularly for other groups (e.g. Bryum ). Ultimately matK sequencing was only attempted for 477 samples due to time constraints and the apparent limitations of the protocols in some lineages. However, of those attempted 388 were sequenced successfully (81%), giving the region quite a high (if somewhat artificial) success rate. For the chloroplast intergenic spacer region trnH-psbA sequencing success was also high at 83%.

9 The ITS2 intergenic spacer was the only barcoding region used from the nuclear genome and this makes it more prone to problems such as fungal contamination, particularly for some liverwort genera which contain fungal symbionts such as Cephaloziella and Riccia . Sequencing success for this region was still relatively high at 75%.

4.2 SPECIES ACCOUNTS

4.2.1 Atrichum angustatum (Brid.) Bruch & Schimp. Taxonomic category: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species.

Atrichum angustatum is a fairly common moss species in both continental Europe and North America, but appears to be in decline in Britain. While historical records exist for Wales, Scotland and Ireland, the majority of the more modern records of the species are from the southeast of England, although it has not been recorded from any sites in Atrichum angustatum in Portugal. Photo: D.G. Long that part of the country for over 20 years. Only one sample of putative A. angustatum from the UK was successfully sequenced for this study (collected by R.V. Lansdown in East Gloucestershire in 2011) as no older herbarium material was suitable for DNA extraction and searches of historic sites in Kent and Sussex were unsuccessful. European and American samples were used in lieu of additional samples from the UK. The DNA barcoding data were used to test whether A. angustatum was genetically distinct from other British Atrichum species, in particular A. undulatum.

DNA Barcoding data Sequencing of this group was generally successful, although of a total of 17 samples 2 failed to amplify for trnH-psbA and 8 for matK . Despite this a sufficient number of samples of the target species and its closest relative in the UK ( A. undulatum ) were available for the results of each barcoding region to be significant. A single accession of the southern hemisphere species A. androgynum was originally included in the analysis as this species has recently been reported from Portugal and concerns were raised that some samples sequenced as A. angustatum might in fact be this species. However, ITS2 data showed the two species to be distinct and the A.

10 androgynum sample was excluded from the tree-based analysis because it failed to amplify for either matK or trnH-psbA and this missing data distorted the tree topology. Figure 1 shows a maximum parsimony tree based on all 4 DNA barcode regions to illustrate the relationships among samples. This analysis resolves each of the ingroup taxa with moderate to strong support and shows no significant variation within each species. The recently collected sample of putative A. angustatum from East Gloucestershire nests within the clade of European and North American samples of A. angustatum , confirming its identification as the only recent record from Britain.

Taxonomic implications The DNA barcode data support the effective species limits as currently recognised (e.g. Smith, 2004) and therefore no taxonomic changes are proposed.

Identification of A. angustatum The most reliable character to distinguish A. angustatum is the smaller leaf cell size, 12-18(-20) µm wide in mid-leaf, compared to 20-40 µm wide in A. undulatum . Mature spores are also smaller (12-14 µm compared to 16-20 µm in A. undulatum ), although this is a less useful character as sporophytes aren’t always present. Smith (2004) suggests that A. angustatum can be distinguished from A. undulatum in the field by the narrower leaves (<1 mm) and occasional presence of a reddish pigmentation, but these characters are unreliable so samples should always be checked microscopically. Some specimens aren’t easily assigned to a species and this suggests that the currently used characters aren’t always sufficient. In cases of ambiguity, e.g. where only depauperate material is available, any of the four DNA barcoding regions used here will resolve the identification of British Atrichum species.

Conservation status of A. angustatum These data prove that A. angustatum is a distinct species in need of conservation action and is particularly at risk given its rarity. The marked decline of this species in Britain, with only one confirmed record in over 20 years is puzzling, particularly considering its relative abundance in continental Europe and North America. Possible reasons for its decline in Britain include changes in land management practices and climate change. It is possible that the species has been overlooked in recent years, although dedicated searches at several historical sites during the course of this project were unsuccessful, and it is perhaps more likely that the species was over-recorded in the past as many records for the species were based on field observations.

Further research • Additional surveying of historic sites. • Research to determine the cause of decline in the UK. • Comparison with other non-UK species.

Fig. 1 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 16 samples of Atrichum with 2 samples of Oligotrichum hercynicum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The tree resolves A. angustatum with moderate bootstrap support, showing it to be a distinct species with a strongly supported sister species relationship to A. undulatum .

12 4.2.2 Barbilophozia kunzeana (Huebener) K. Müller Taxonomic category: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species.

This species has been recorded from scattered bog sites across Wales, northern England and Scotland. As well as similarities to other congeneric species, B. kunzeana can be confused with species of Lophozia and Tritomaria . It can be difficult to find in the field as it tends to grow as scattered shoots through other plants. Barbilop hozia kunzeana . Photo: D.G. Long The species has also been reported growing on rock which is a significant ecological difference worthy of investigation but unfortunately no suitable material from this habitat was available for sequencing in this study. Lab staff at RBGE had already sequenced several samples of species related to B. kunzeana as part of another project and these samples were also available for use in this analysis. The DNA barcoding data were used to test whether B. kunzeana is distinct from other morphologically similar species in the UK.

DNA Barcoding data 51 samples of Barbilophozia , Lophozia and Tritomaria were included in the analysis. 4 samples failed to amplify for rbcL , 8 for matK , 6 for trnH-psbA and 10 samples for ITS2. Figure 2 shows the maximum parsimony strict consensus tree for the combined dataset of all four barcoding regions with B. kunzeana distinguished from all other species with strong bootstrap support (100%). The additional variation within this clade results from two base substitutions in trnH-psbA which appear to be random mutations and are not deemed to be taxonomically significant.

Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Paton, 1999) and therefore no taxonomic changes are proposed.

Identification of Barbilophozia kunzeana The characters described in Paton (1999) are the most reliable for identification of this species, with the bilobed lateral leaves distinguishing B. kunzeana from other Barbilophozia spp. and the well-developed bifid underleaves distinguishing it from other liverworts with bilobed leaves.

13 In cases where morphological identification is not possible (e.g. atypical or depauperate material) any of the four barcoding regions used here will distinguish B. kunzeana from the other species studied.

Conservation status of Barbilophozia kunzeana The barcode data show B. kunzeana to be a genetically well-defined species and therefore current conservation efforts should be maintained. Because it can be easily overlooked in the field and there is no shortage of apparently suitable habitat Paton (1999) suggested that it may be more frequent than records suggest. While this is undoubtedly true and the species is still being found at new sites, it is still extremely rare and is also being lost from some historical sites due to habitat degradation.

Further research • Cryopreservation and reintroduction programmes to historic sites. • Sequencing of material from rock habitats for comparison with the more common bog populations.

Fig. 2 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 51 samples of Barbilophozia and related species, with 1 sample of Cephaloziella hampeana as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves B. kunzeana as a distinct species with strong bootstrap support. The intraspecific variation within this clade is the result of a 2 base substitution in the trnH-psbA spacer region and does not appear to be taxonomically significant.

15 4.2.3 Bryum spp. Taxonomic categories: 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus; 4) Species which are problematic due to uncertainty over their distinctness and generic placement.

Bryum is the most speciose bryophyte genus in Britain and is notoriously difficult taxonomically. As well as substantial problems with identification and determining species boundaries in Bryum , the generic placement of many species is open to debate, with mounting evidence that the generic classification of the Bryaceae family as a whole needs to be revised. Six problem threatened species were included in the study: B. gemmiparum , B. knowltonii , B. salinum , B. warneum , B. calophyllum and B. marratii , with the aim of studying the genetic variation within each species and also determining whether they are distinct from related taxa.

The species accounts for Bryum which follow have been prepared by Bryaceae expert Dr D.T. Holyoak.

DNA Barcoding data Sequencing was relatively successful for rbcL , trnH-psbA and ITS2 but matK was particularly difficult to amplify in this group. The reasons for this have not yet been identified but it may be that the primer pairs being used are not specific enough for the genus. A 9 base pair inversion was observed in trnH-psbA which can generate a strong misleading phylogenetic signal and lead to incongruent results, so these were optimised by inserting gaps in the sequence alignment to separate the inversion types. This inversion doesn’t appear to have any phylogenetic significance and occurred randomly throughout the dataset with some species in the dataset represented by samples both with and without the inversion but otherwise identical in their sequences. Species resolution was relatively poor for rbcL and trnH-psbA , while ITS2 showed a much more promising degree of variability but the sequences proved difficult to align unambiguously so many potentially informative bases had to be excluded from the analysis. The strict consensus tree from a maximum parsimony analysis of combined data from the 3 successfully sequenced regions (figure 3) is not very informative in terms of the deep phylogenetic relationships amongst taxa and this limited observation on generic placement. However, the data did resolve several key species with moderate to strong support and indicates how some of these relate to one another. Of the target priority species B. calophyllum , B. marratii , B. knowltonii and B. warneum were all resolved with strong support, with some additional variation noted in B. knowltonii . The two samples of B. salinum are resolved with relatively low support, although sequences for the two samples are identical with the exception of the inversion in trnH-psbA being present in one sample but not the other. Most samples of B. gemmiparum included in the analysis had more or less identical sequences (with the exception of the trnH-psbA inversion present in some samples) and were resolved in a clade with moderate support but one sample from Devon (B. cf. gemmiparum 10-02827 in fig. 3) showed more variation and appears to have a closer affiliation to the B. dichotomum complex of species.

16 Bryum marratii Hook.f. & Wilson

Taxonomic implications These are the first DNA sequence data to be obtained for B. marratii and therefore valuable in confirming its status as a species distinct from several other European species that are likely to be among its close relatives. Preliminary cladograms suggest it may not belong in (sub)genus Ptychostomum as delimited by Holyoak & Pedersen (2007) but Bryum marratii in Ireland. Photo: D.T. Holyoak further analyses including additional species (especially Bryum uliginosum (Brid.) Bruch & Schimp, the type of Ptychostomum ) are needed to test this.

Identification of Bryum marratii The gametophytes of B. marratii and B. calophyllum can be difficult to distinguish when the plants are small. Their capsules are more distinctive, but these are infrequently produced, especially in B. marratii . The DNA sequence data are useful in confirming that distinct species are involved and that good non-fertile material of B. marratii is being identified consistently.

Conservation status of Bryum marratii This species mainly inhabits coastal habitats in the Boreal zone, where it is known mostly from Europe, with only three confirmed records elsewhere (in N. America). Although listed as 'Not threatened' for Europe as a whole, this should be reassessed since Ireland and Sweden may now be the only countries with numerous extant populations. Elsewhere on the continent it has one site in north-western France and several on coasts of the Netherlands. It was always very rare in Germany and is now absent from numerous former localities (with post-1960 records from three coastal sites and one inland). There are apparently few or very few records from all other countries in its range (Iceland, Norway, Denmark, Finland, Estonia, Latvia, Poland, Slovakia, Canada and U.S.A.). Following an apparent decline it is now rare in Britain, so listed as 'Endangered' by Church et al. (2001) and 'Vulnerable' in a revised list using IUCN criteria by Hodgetts (2011). Records from the last decade are from two localities in England (small populations in Cumbria and N. Northumberland), three in Wales and at least five in Scotland. Nevertheless, B. marratii may be overlooked to some extent because the usual habitat for it at the upper edge of salt marshes receives less attention from bryologists than places with richer bryophyte floras. In Ireland and doubtless elsewhere suitable habitat is declining in extent because of reduction of grazing on salt marsh edges.

17 Bryum calophyllum R.Br.

Taxonomic implications These are the first DNA sequence data to be obtained for B. calophyllum and thus they are valuable in supporting its status as a species distinct from others known in Britain. Further studies are needed to clarify the relationship to B. calophyllum of B. axel- blyttii Kaur. ex H.Philib. and B. acutiforme Limpr. in I.Hagen (treated as Bryum calophyllum in Ireland. Photo: D.T. Holyoak synonyms by Holyoak 2004) and numerous other nominal taxa from Scandinavia and the Arctic (all of them described in detail as components of Subsect. Calophylloidea Kindb. by Podp ěra 1973).

Identification of Bryum calophyllum As discussed above, the main difficulty in identifying B. calophyllum arises from similarity of small non-fertile plants of this species to those of B. marratii . The DNA sequence data are useful in confirming that identification based on gametophytic characters is being achieved successfully with well-grown material. See notes under B. warneum below for discussion of a gathering from Scotland that seems to combine some characters of B. calophyllum with those of that species.

Conservation status of Bryum calophyllum B. calophyllum appears to be commonest in the Arctic. It occurs throughout the Canadian Arctic, but more often in the high Arctic, and it is not rare in Iceland. Hence it seems that the species is not globally threatened. Nevertheless, it is listed as 'Rare' in Europe as a whole (Schumacker & Martiny 1995), where it has shown marked declines towards its southern range limits. In Britain it is very local and undoubtedly declining, so listed as 'Vulnerable' by both Church et al. (2001) and Hodgetts (2011). There are recent records at only one site in Wales, none in England, but several in Scotland; loss or degradation of its habitat in open dune-slacks has apparently caused the declines.

18 Bryum warneum (Röhl.) Brid.

Taxonomic implications These are the first molecular data on B. warneum and therefore valuable in supporting its status as a distinct species from other Bryaceae occurring in the British Isles, including the morphologically rather similar B. arcticum (which is mainly montane outside the Arctic, but recorded from coasts at least in the Aland Is.: DTH Bryum warneum capsules, Sefton Coast, Merseyside. unpublished). Photo: D.T. Holyoak Provisional phylograms imply it may belong in (sub)genus Ptychostomum as delimited by Holyoak & Pedersen (2007), tending to confirm the judgment of Spence (2009) based on morphological characters. B. warneum is usually described as autoicous in the literature, although recent studies (Holyoak 2001) show that a few populations are synoicous but otherwise apparently similar in morphology and ecology. The present study compared a synoicous population (Dungeness, v.c. 15) with two that are autoicous (v.c. 59, v.c. H21), demonstrating that they belong in the same clade. Comparable intraspecific variation in sexuality is known in a few other Bryum , notably B. torquescens Bruch & Schimp. However, none of the other species is known to have clearcut differences in sexuality of whole local populations. In B. warneum this might result from "founder effects" at its widely isolated coastal colonies. One sample of B. warneum used in this study (E00619687: D.G. Long 31606, In salt marsh, East Lothian: John Muir Country Park) produced sequence data suggesting a position somewhat isolated from the clade demonstrated by the other three populations sampled. Detailed re-examination of the voucher specimen confirms that it shows characters expected of B. warneum which would lead to it being keyed out as that species in the standard floras (leaves ovate, acute at apex, lacking red at base, bordered, with shortly excurrent costa; exostome teeth with oblique cross-walls; endostome cilia rudimentary; spores well formed, 34-42 µm, mostly ca 40 µm). Nevertheless, the few capsules available appear short for B. warneum and not obviously narrow-mouthed as typical in that species (see photo) and the exostome teeth are rather short (430-440 µm) and perforated along the midline. The capsules were also old and dehisced on 15th September 2002, whereas capsules of B. warneum typically mature later (October-November). Those differences from B. warneum strongly suggest some of the characters of B. calophyllum , which is among the few congeners from coastal habitats with similarly large spores. Acute-leaved forms of B. calophyllum occur in Scandinavia, where as noted above, they are sometimes separated as the rare species B. axel-blyttii Kaur. ex H.Philib. and B. acutiforme

19 Limpr. in I.Hagen (treated as synonyms of B. calophyllum by Holyoak 2004). Since morphology and DNA sequences suggest affinity of Long 31606 to both B. warneum and B. calophyllum , further investigation of this population is desirable to assess whether it may represent a population that has arisen from interspecific hybridisation. If so, it might shed light on the status of B. axel-blyttii and other puzzling forms. An American specimen distributed as Imbribryum mildeanum (COLO (Boulder) B- 117614, in Herb. DTH, collected Chaffee Co., Colorado at 12300 ft altitude) gave surprising DNA sequence data suggesting it is closer to B. warneum than any of the other species studied. The specimen consists of small cushions of rather featureless, non-fertile, small-leaved plants that do not show the characters of B. mildeanum (the stems and leaf bases are not red; leaves are ovate, acute, concave) but they could be dwarfed B. warneum . The DNA sequence data apparently discount B. arcticum as a possible identification. Since B. warneum is currently accepted as occurring in N. America only at a single coastal locality in Quebec (Shaw & Marcotte 1983, Spence 2009) it is clear that further study is needed to clarify the taxonomic status of this population.

Identification of Bryum warneum Fertile specimens with mature capsules and spores are needed for secure identification of Bryum warneum , which is usually unproblematic if good material is carefully studied. The most similar species is B. arcticum , which is rather variable, but always synoicous (never autoicous) and has smaller spores ((20) 25-36 (39) µm); other differences are its longer, narrower and somewhat asymmetrical capsules that are horizontal or inclined to cernuous (not pendulous). The present study establishes that an atypical synoicous population at Dungeness, Kent is B. warneum (with spores 33- 37 (41) µm and typical capsules for this species).

Conservation status of Bryum warneum B. warneum was listed as 'Rare' for Europe as a whole (Schumacker & Martiny 1995). It is rare in Britain, where lost from some localities in England due to coastal development. Church et al. (2001) listed it as 'Vulnerable', which was revised to 'Near Threatened' using IUCN criteria by Hodgetts (2011). Records since 2000 are from only four localities in Wales and four in England (Holyoak 2002a, 2002b, and unpublished), but there are more sites in Scotland.

20 Bryum knowltonii Barnes

Taxonomic implications These are the first DNA sequence data to be reported for B. knowltonii , so it is significant to confirm that it appears to be a valid species with respect to other British Bryum . Samples from three populations of B. knowltonii form a clade distinct from a larger group of relatives comprising B. archangelicum , B. salinum , B. intermedium Gametophytes of Bryum knowltonii , Holy Island, Northumberland. and B. algovicum , all of Photo: D.T. Holyoak which also share numerous morphological characters with B. knowltonii . The provisional phylograms imply it belongs in (sub)genus Ptychostomum as delimited by Holyoak & Pedersen (2007), tending to confirm the judgment of Spence (2009) estimated from morphological characters. Inclusion of a Belgian sample (Dirk De Beer no. 2014, from Antwerp harbour) among those sequenced is valuable because that specimen has very large spores (33-43 µm), as in a few other European populations (Holyoak 2004), far larger than the 20-30 µm given in various recent British and European floras. Since the sample forms part of the same clade as specimens from Norfolk (spores 26-36 µm) and Northumberland (spores 18-23.5 µm) it seems clear that spore size is more variable in the species than was described in older accounts, without any need to invoke hybridisation with large- spored species such as B. warneum . It would be interesting to have chromosome counts from small- and large-spored populations to check for possible polyploidy in the species, for which only n = 11 has so far been reported.

Identification of Bryum knowltonii Only fertile material of B. knowltonii with mature capsules can be identified with certainty, although the weakly bordered leaves with costa percurrent rather than excurrent exclude some related species. In the field, the pyriform capsules and tall seta suggest B. warneum , although the capsules are usually shorter and microscopic study of the plants soon reveals the red leaf bases in B. knowltonii . As noted in the preceding section, the molecular data confirm that a spore size range of 18-43 µm can occur in B. knowltonii , far exceeding the 20-26 µm given by Smith (2004). Distinction of B. knowltonii from B. archangelicum and B. salinum requires good material and particular care: it is best based on the latter two species having a more longly excurrent costa, better defined leaf border and a longer capsule with longer neck.

21 Conservation status of Bryum knowltonii Listed as 'Not threatened' for Europe as a whole. Following a large decline, now listed in Britain as 'Vulnerable' (Church et al. 2001, Hodgetts 2011). Recent British records are mainly from sandy dune-slacks, with a large population also persisting on a sandy salt-marsh edge on Holy Island (N. Northumberland). Older records include some from ruderal habitats, including railway ballast.

Bryum salinum I.Hagen ex Limpr.

Taxonomic implications These are the first DNA sequence data obtained from B. salinum and therefore important in supporting its status as a species distinct from B. archangelicum , although data from more populations of both taxa are needed for this to be securely established. Because of the strong morphological similarity to B. archangelicum the position of B. salinum in (sub)genus Ptychostomum has already been presumed (Spence 2009), but now it is confirmed.

Identification of Bryum salinum Reliable separation from commoner coastal Bryaceae species depends on characters of the mature capsule. B. salinum is most similar to B. archangelicum , from which it differs in the capsule mouth when mature being red not yellowish, the exostome teeth often being perforated (cf. no perforations), endostome processes with fine uniform papillae (cf. more strongly papillose down middle than at edges), exostome teeth straw coloured (cf. yellow above and reddish below), larger exothecial cells, and neck of capsule gradually (cf. abruptly) tapered into seta; B. archangelicum also has less porose leaf cells. The present study revealed a hitherto overlooked population of B. salinum at Holy Island, N. Northumberland, providing the third record of it from England and only the second extant population. This was based on the specimen D.G. Long 39594 from sand at edge of saltmarsh at grid reference NU 1059 4340 collected on 15th August 2010, a poor specimen with only few and rather old capsules. It was tentatively identified as B. salinum by DGL and forwarded to DTH who thought it was more likely to be B. knowltonii , mainly on the basis of the rather short capsules and only shortly excurrent costae on some leaves. Furthermore, that species was already well known at the same site and it had been collected within 10 metres of the location in the same habitat (D.T. Holyoak 01-987, from NU 1059 4341, collected 28th September 2001, along with more material collected in 2008). Nevertheless, the new DNA sequence data reveals that Long 39594 forms a clade with an undoubted specimen of B. salinum from Cumbria, whereas Holyoak 01-987 clearly clusters with B. knowltonii from elsewhere, besides showing appropriate morphology for that species. Subsequent re-examination of Long 39594 shows that the costa is clearly excurrent in many leaves, so that it is unlikely to be B. knowltonii ; it shows other characters of B. salinum rather than the commoner B. archangelicum in conformity with the DNA evidence. Re-examination of other gatherings from the same locality (notably Holyoak 08-677 from NU 10589 43404 collected 7th November 2008) suggests non-fertile plants with an excurrent costa occur mixed amongst B. knowltonii . Thus, B. salinum appears to have been overlooked here because it bears few if any capsules; also, the capsules ripen in the summer, so that they are old and easily missed on autumn visits.

22 B. salinum must be somewhat overlooked elswhere since microscopical study of material with ripe capsules is necessary for identification. Non-fertile and apparently unidentifiable Bryaceae that might be this species or one of its commoner relatives are frequently encountered at upper edges of salt-marshes, at least in N. Britain and N. Ireland. Patient searching around mid- summer for good fertile material in this bryologically somewhat neglected habitat may well be rewarded with more records of B. salinum . Hence, in future, barcode data may offer a powerful tool for checking records of B. salinum , since sporophytes are not needed. The habitat disturbed by tidal flooding and grazing around salt-marsh high-tide lines coupled with sparse or non- existent capsule production currently makes the species very difficult to find, even with targeted surveys. Capsule of Bryum salinum , Sweden. Photo: T. Hallingbäck Conservation status of Bryum salinum The latest British bryophyte Red List compiled using IUCN criteria classifies B. salinum as 'Endangered' (Hodgetts 2011). Nevertheless, a 'Data Deficient' classification might be more appropriate, in view of the difficulties in surveying the species described above. Whereas it is almost certainly a rare or very rare plant in southern Britain, the impossibility of identifying non-fertile material even with microscopic study may obscure its status in N. England and Scotland.

Bryum gemmiparum De Not.

Taxonomic implications DNA sequences from two specimens of B. gemmiparum from the Canary Islands were obtained by Pedersen et al. (2007), showing that the (sub)generic affinities of the species are with Bryum s.s. (Holyoak & Pedersen 2004), rather than Imbribryum ( B. alpinum , B. mildeanum ) as previously assumed from morphological evidence. The present data Bryum gemmiparum with bulbils, Ireland. Photo: D.T. Holyoak

23 from a wider range of geographical populations (SW. England, S. Wales, Ireland, Canary Islands) support B. gemmiparum as a distinct species from other British Bryaceae, although one sample identified as B. gemmiparum rests outside this clade within the B. dichotomum complex.

Identification of Bryum gemmiparum Studies on herbarium material for a "Guide to European Bryaceae" (Holyoak in prep.) have led to a reappraisal of the characters by which B. gemmiparum should be identified. In particular, it appears that presence of spherical rhizoidal gemmae (A.C. Crundwell in Hill et al. 1994, Smith 2004), far from characterising B. gemmiparum , indicates that another species is involved (usually an atypical form of B. alpinum ). The following text prepared for the "Guide" summarises the revised identification criteria for the species:

"B. gemmiparum is intermediate in leaf form and areolation between typical B. alpinum and B. muehlenbeckii, with both leaves and mid-leaf cells proportionately wider than in the former but narrower than in the latter. It is clearly distinct from both in the occurrence of large axillary bulbils and needs to be determined with care (if at all) when these are lacking, narrow leaved forms of B. gemmiparum being particularly troublesome to determine. The shortly acute and often concave leaves of B. gemmiparum differ from the tapering leaves with flat apices normal in B. alpinum, but many specimens placed as B. gemmiparum in herbaria are nevertheless forms of B. alpinum with wide leaves. Presence of reddish tubers in the tomentum of rhizoids often confirms that B. alpinum is involved, since tubers appear to be lacking in B. gemmiparum. Early stages of branch development in B. alpinum can give a misleading impression that axillary bulbils are present, although these are not as numerous as the bulbils in B. gemmiparum and they elongate before becoming as large." "Robust plants of B. dichotomum have sometimes been confused with B. gemmiparum, but these have a weaker costa (rarely >60 µm wide at leaf base, cf. >80 µm in B. gemmiparum), relatively wider ± hexagonal mid-leaf cells, and the forms with costa evanescent or percurrent usually also differ in leaf shape."

A specimen from Madeira (L. Luis 55, from Herb. DTH) named as B. mildeanum by DTH in 2007 produced DNA sequence data that clearly place it in the same clade as B. gemmiparum . Re-examination of this specimen shows that it cannot be B. mildeanum (largest leaves with costa wide and strong, up to 85 µm wide at leaf base; costa percurrent not excurrent; leaf base and stem not red; upper part of leaf too wide, less tapered than in B. mildeanum ). B. gemmiparum is known by better material from other rivers on Madeira, whereas B. mildeanum may not have any correct records from the island. Nevertheless, on morphological criteria alone the identification of LL 55 as B. gemmiparum remains tentative: the leaves are scarcely concave and no axillary bulbils are present. Tiny brown "tubers" of a few cells on the ends of some of its rhizoids appear to be galls induced by fungi (cf. Martínez-Abaigar et al. 2005). LL 55 perhaps represents shaded plants.

Conservation status of Bryum gemmiparum Listed as 'Not threatened' for Europe as a whole, since it is locally common in parts of southern Europe (e.g. listed as 'Not threatened' for both Spain and Portugal). It is much scarcer around its northern range limits: Rare and apparently declining in Britain, so listed as 'Endangered' by Church et al. (2001) and Hodgetts (2011). Recently discovered in Ireland, where rare and included in the Red List and regarded as Critically Endangered in Switzerland.

Fig. 3 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, trnH-psbA & ITS2 for 57 samples of Bryum with 1 sample of Ulota phyllantha as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The target priority species are generally resolved with moderate to strong support although B. warneum and B. gemmiparum each have one sample nesting elsewhere in the tree.

25 4.2.4 Cephaloziella spp. Taxonomic categories: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

Cephaloziella is a genus of diminutive leafy liverworts which can be very difficult to identify (often impossible when sterile) due to their size and there are few reliable characters to distinguish some species. The genus includes four UK priority species ( C. baumgartneri , C. calyculata , C. integerrima and C. nicholsonii ) and another Cephaloziella nicholsonii . 9 species accepted in the British flora. Photo: D.A. Callaghan Samples of each of the target priority species except C. baumgartneri were available for analysis. Species of Cephaloziella are taxonomically critical and this study aimed to use DNA barcoding markers to test species boundaries within the genus in Britain.

DNA Barcoding data Sequence data was successfully obtained for 37 samples of Cephaloziella , with only one sample failing to amplify for rbcL , two failing for matK and one for trnH-psbA . Only 13 samples (35%) amplified successfully for ITS2 and this may be due to the presence of fungal symbionts in Cephaloziella , which can interfere with PCR of regions in the nuclear genome. Despite the success of sequence amplification in the chloroplast barcoding regions the resulting analysis was not very informative. Figure 4 shows a strict consensus maximum parsimony tree based on data from all four barcoding regions, however any structure in the tree topology does not support the morphologically determinations, with taxon names seemingly scattered on the tree.

Taxonomic implications These data suggest that the characters currently used (e.g. Paton, 1999) to distinguish between species of Cephaloziella are inadequate. Although some of the anomalies in the results may be due to misidentification of samples or the wrong stems being extracted from mixed collections, it is likely that there are also more serious issues and the group as a whole needs urgent taxonomic attention. However, the well- supported basal clade containing C. calyculata, C. turneri and two samples of C. integerrima appears to show the true relationships of these three species which are more distinct morphologically than the other species of the genus. This group was added to the project relatively late and specimens have not been revisited due to time constraints. However, reassessing the morphological characters

26 of these samples in light of the molecular results may help to resolve some of these issues. Additional sampling of under-represented taxa would also be desirable.

Identification of Cephaloziella spp. The morphological characters currently used for identification of this group are shown to be inadequate and major taxonomic revision is required to resolve this.

Conservation status of Cephaloziella spp. For a reliable conservation assessment this group requires urgent taxonomic revision.

Further research • Full taxonomic revision using both morphological and molecular data. • Sequencing ITS2 by overcoming problems caused by fungal symbionts, e.g. by culturing from spores or cloning.

27 Fig. 4 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 37 samples of Cephaloziella with 1 samples of Nowellia curvifolia as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. Although the basal clade is well resolved, the limited structure in the rest of the tree does not support the morphological determinations and suggests that the group requires further study.

28 4.2.5 Ceratodon conicus (Hampe) Lindb. Taxonomic category: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species.

C. conicus is a very rare moss species which appears to be declining in Britain due to habitat destruction, particularly the decline of mud-capped walls (Porley & Hodgetts, 2005). It differs from its ubiquitous relative C. purpureus (pictured) primarily in sporophyte characters and habitat, preferring more calcareous substrates than C. purpureus . This study used DNA barcoding markers to test whether C. conicus is genetically distinct Ceratodon purpureus . Photo: M. Lüth from C. purpureus .

DNA barcoding data Only one herbarium specimen, lacking sporophytes, of putative C. conicus suitable for DNA extraction (sent from West Gloucestershire by P. Martin) was available for the study and its apparent decline meant that fresh sampling was unfeasible. Because of the importance of sporophytes for accurate identification the available sample could not be confidently assigned to C. conicus based on morphology, and initial difficulties in successfully sequencing the sample for all regions except rbcL meant that the results were inconclusive. However, the trnH-psbA barcoding region was eventually sequenced successfully and this along with rbcL showed sufficient variation compared to five widely distributed C. purpureus samples for a more confident assessment. The Gloucestershire sample differed from the five samples of C. purpureus by 1 base substitution in rbcL and 3 substitutions and 1 indel in trnH- psbA .

Taxonomic implications The barcoding data imply that the Gloucestershire specimen is a distinct taxon from C. purpureus and the excurrent nerve and calcareous substrate suggest that it is referable to C. conicus , thus supporting its status as a distinct species rather than a habitat form of C. purpureus . However, additional sampling (including foreign material) and additional efforts to sequence older samples of C. conicus would be desirable to test the consistency of the genetic variation seen here.

Identification of Ceratodon conicus Sporophytes of C. conicus differ from those of C. purpureus in being held erect and having unbordered peristome teeth (Smith, 2004). Without sporophytes the two species can be difficult to distinguish due to the variable nature of C. purpureus , although plants growing on a calcareous substrate with leaves with a longly excurrent nerve are good candidates for C. conicus .

29 These results suggest that rbcL and trnH-psbA are both effective molecular markers for confirming the identity of sterile or depauperate material suspected of representing C. purpureus .

Conservation status of Ceratodon conicus This data suggests that C. conicus is a distinct species and due to its rarity should be a conservation priority. Although the species appears to have declined to near extinction the lack of reliable vegetative characters to distinguish it from the very common C. purpureus might mean that it is being overlooked and additional efforts to relocate it at historical sites would be worthwhile given the potential for DNA barcoding to confirm the identification of sterile plants.

Further research • Additional surveying of historic sites. • Research to determine the cause of decline in Britain. • Sequencing from European and North American populations for comparison. • Design ‘minibarcodes’ to attempt sequencing of older fertile specimens. • Further efforts to generate sequence data for matK and ITS2 barcode regions.

Fig. 5 Maximum Parsimony strict consensus tree for the combined dataset of rbcL & trnH-psbA for 6 samples of Ceratodon with 1 sample of Dicranella palustris as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The single sample referable to C. conicus is shown to be distinct from 5 broadly distributed samples of C. purpureus which had identical sequences across the regions studied.

31 4.2.6 Ditrichum Ditrichum cornubicum Paton Taxonomic categories: 2) Species which may consist of 2 or more distinct cryptic species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

Ditrichum cornubicum is a very rare moss restricted to spoil from old copper mines. It was described as a new species relatively recently (Paton, 1976) and is only known globally from two sites in Cornwall and one in the west of Ireland. Ditrichum cornubicum . Photo: D.A. Callaghan

Ditrichum plumbicola Crundw. Taxonomic categories: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

Ditrichum plumbicola is another metalliferous species restricted to lead mine spoil at scattered sites throughout the UK and Ireland. Since its discovery and description as a new species based on British samples (Crundwell, 1976) it has also been recorded from Germany and Belgium. Frahm et al. (2008) placed it in Ditrichum plumbicola . Photo: D.A. Callaghan the synonymy of D. lineare based on molecular results from a single cpDNA region which did not resolve the two species as reciprocally monophyletic, but this has since been rebuked by Sotiaux & Vanderpoorten (2011) who argue that recently diverged taxa will not necessarily be reciprocally monophyletic.

32 This study aimed to test species boundaries within Ditrichum , particularly between D. cornubicum , D. plumbicola and D. lineare , and also to look for intraspecific variation in these species.

DNA Barcoding data All 5 samples of D. cornubicum available were sequenced successfully for all four barcoding regions, with the exception of one sample failing in matK . Of 14 available samples of D. plumbicola 4 failed across all barcoding regions with an additional sample failing for ITS2 and 6 failing for matK . In ITS2 all samples of D. cornubicum were identical, but they varied so much from the D. lineare and D. plumbicola samples that the data were impossible to align satisfactorily and therefore a tree based analysis using the ITS2 data could not be performed. D. plumbicola and D. lineare were distinguishable in ITS2 by 6 substitutions and 3 sets of indels and were readily alignable. The maximum parsimony strict consensus tree based on data from the three chloroplast barcoding regions is shown in figure 6. The data resolve both D. cornubicum and D. plumbicola as distinct and conserved species with strong bootstrap support. D. plumbicola is resolved as a sister species to D. lineare as would be expected from their morphological similarities. Two samples originally identified as D. plumbicola were flagged as misidentifications of D. lineare and another (along with two samples originally identified as D. lineare ) appears to be a misidentified species of Dicranella . These last three samples remain uncorrected on the tree, as their true identity has not yet been satisfactorily resolved.

Taxonomic implications D. cornubicum has been shown to be a distinct and well-supported species and no additional genetic variation was found within the samples to support the hypothesis of cryptic species being present. However, no material from the disjunct Irish population in West Cork was available for this study to further test this hypothesis. D. plumbicola has also been shown to be a distinct and conserved species, although the morphological characters used to identify it appear to be unreliable, particularly with respect to D. lineare . This analysis of three chloroplast regions also resolves D. plumbicola and D. lineare as reciprocally monophyletic, which would satisfy the species concept of Frahm et al. (2008) and contradicts their earlier synonymy of D. plumbicola under D. lineare .

Identification of Ditrichum cornubicum & D. plumbicola Both species are most likely to be confused with D. lineare but the more spreading leaves with shorter, broader cells and the presence of rhizoidal gemmae are diagnostic for D. cornubicum . D. plumbicola is much closer to D. lineare . It can normally be distinguished by the shorter leaves (2-3 times longer than wide) and plane leaf margins, which are recurved on one or both sides in D. lineare . Another aid to identification is habitat, with D. cornubicum restricted to old copper mine spoil and D. plumbicola restricted to lead mines.

33 Conservation status of Ditrichum cornubicum & D. plumbicola D. cornubicum is extremely rare, known to be extant at only three sites globally (two in Cornwall and one in Ireland; the plant has been lost from its original site in Cornwall). All three populations are on protected land but the species is still potentially vulnerable to threats such as disturbance by humans or animals, shading from successional vegetation and eutrophication from sheep (Lockhart et al. 2012). D. plumbicola is more widespread with scattered populations in Britain, Ireland, Germany and Belgium. It faces similar threats to D. cornubicum .

Further research • Generate sequence data from the Irish population of D. cornubicum . • Comparison with European populations of D. plumbicola as Frahm et al. (2008) reported that ITS sequences of D. plumbicola and D. lineare were unalignable, which was not found to be the case with British material. • Further sequencing of Ditrichum and Dicranella to investigate issues of generic placement.

Fig. 6 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK & trnH-psbA for 40 samples of Ditrichum and relatives with 1 sample of Ceratodon purpureus as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. This analysis resolves both priority species in strongly supported clades with no intraspecific variation shown, reinforcing their treatment as distinct and conserved species in need of conservation.

35 4.2.7 Ephemerum cohaerens (Hedw.) Hampe 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

This is a tiny ephemeral moss species growing in inundation zones of reservoirs. Insufficient herbarium material was available for the target species and targeted collecting was unfeasible due to the transient nature of the plants, meaning no analysis could be undertaken for this species. As an ephemeral species the spores are presumably persistent and culturing of these from herbarium material may offer a solution to sampling difficulties, as well as problems with contamination likely to be encountered with such diminutive plants growing in this type of habitat.

Further research • Culturing from spores to obtain sufficient pure material for DNA extraction.

4.2.8 Eurhynchiastrum pulchellum (Hedw.) Ignatov & Huttunen 2) Species which may consist of 2 or more distinct cryptic species.

This pleurocarpous moss has been recorded from crumbling basalt at a handful of upland sites in Scotland and Ireland, while at its only site in England it was recorded in an entirely different habitat from an area of exposed chalk in the lowlands of Suffolk. Sanford & Fisk (2010) report that the plant has not been seen in Suffolk Eurhynchiastrum pulchellum . Photo: D. Bell since 1980 and that the species has probably been lost from there as the site where it had been recorded has become grassed over. Three varieties of E. pulchellum are recognised in Europe but Hill (1993) referred all British and Irish material to var. diversifolium . This study aimed to determine the level of genetic variation between UK populations, particularly assessing whether plants growing in the two very different habitats represent distinct cryptic taxa.

DNA Barcoding data Sequencing was generally successful for rbcL , trnH-psbA and ITS2, but matK data was not generated due to time constraints and initial failures. Several foreign samples

36 of E. pulchellum were included to enhance the analysis and put the British material in a broader context. In order to test the hypothesis that cryptic species were present at the two contrasting habitats in Britain sequences were required from samples from each habitat. Only one old sample from the calcareous habitat in Suffolk yielded DNA and this was only amplified successfully for the ITS2 barcoding region. An analysis of the rbcL, trnH-psbA & ITS2 data (figure 7) resolved the E. pulchellum samples into two main clades, with all British samples grouping together in a clade which also contained Russian, French and Italian samples. The second clade containing Russian, Czech, French and Estonian samples seems to be referable to E. pulchellum var. pulchellum , although time constraints did not allow closer investigation of these samples as they were beyond the scope of the current study.

Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Smith, 2004) and therefore no taxonomic changes are proposed.

Identification of Eurhynchiastrum pulchellum E. pulchellum var. diversifolium can be distinguished from related taxa by its rounded, often short and julaceous densely-leaved branches and short laminal leaf cells.

Conservation status of Eurhynchiastrum pulchellum This species is extremely rare in Britain, known from only a handful of mountain sites where it is vulnerable to erosion of the dry friable rock it grows on. The population from exposed chalk in Suffolk appears to have been lost (Sanford & Fisk, 2010).

Further research • Additional study of non-UK material as variation seen here suggests the barcode data could shed light on the status of the varieties of E. pulchellum .

Fig. 7 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 22 samples of Eurhynchiastrum & Eurhynchium with 1 sample of Bryum algovicum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolved samples of E. pulchellum from across Europe and Russia into 2 main groups, with all 4 UK samples contained in the larger clade.

38 4.2.9 Leiocolea rutheana (Limpr.) Müll.Frib. 2) Species which may consist of 2 or more distinct cryptic species.

This is a relatively large leafy liverwort growing in rich fens. Two varieties of L. rutheana are currently accepted in Britain (Paton, 1999), with var. laxa endemic to a single extant site in West Norfolk (extinct from its other known sites in Norfolk & Berkshire) and var. rutheana scattered across a handful of sites in northern England, southern Leiocolea rutheana var. rutheana . Photo: D. Bell Scotland and Ireland. This study aimed to assess the level of genetic variation between populations of L. rutheana and consider the status of L. rutheana var. laxa .

DNA Barcoding data 6 samples of L. rutheana var. rutheana were successfully sequenced for 3 of the barcoding regions, with only one of these failing to amplify for matK . Sequence data was obtained for all 4 regions for one sample of L. rutheana var. laxa , although for matK only a partial unidirectional sequence was obtained. A second sample of var. laxa was successfully sequenced for trnH-psbA and ITS2 but this sample was excluded from the combined four region analysis (figure 8) as the missing data from rbcL and matK obscured the tree topology. An analysis of data from all four barcoding regions resolved L. rutheana var. rutheana from all other taxa with strong bootstrap support. All samples of L. rutheana var. rutheana were identical in matK and ITS2, with the specimens from Ireland and vc80 differing by one base substitution in rbcL and 2 in trnH-psbA ; the sample from vc79 shared one of these substitutions in psbA-trnH . L. rutheana var. laxa was resolved in a clade with L. gillmanii , with which it was identical across the regions successfully sequenced. One sample of L. collaris was resolved as sister species to the L. gillmanii /var. laxa clade, while the other L. collaris sample appears to be a misidentification of L. bantriensis with which it is identical across all 4 barcoding regions.

39 Taxonomic implications The barcoding data do not support the treatment of var. laxa as a variety of L. rutheana but suggest a closer affinity with L. gillmanii (under which it has also been treated as a variety in the past). Because var. laxa is morphologically rather intermediate between L. rutheana s.s. and L. gillmanii , Paton (1999) suggested that it might warrant placement at specific rank, but the data presented here suggest it would be more appropriate to treat it as a variety of L. gillmanii , or synonymised with that species. The genetic variation within var. rutheana may warrant further investigation but no morphological discrepancies supporting these genetic differences were noted in this study.

Identification of Leiocolea rutheana Both taxa currently treated as varieties of L. rutheana are monoicous, which will distinguish fertile material from all similar species except L. gillmanii . The decurrent lateral leaf base and large, multiciliate underleaves are diagnostic for L. rutheana var. rutheana . Var. laxa can be differentiated from L. gillmanii by its larger size and arcuate, slightly decurrent leaf insertion.

Conservation status of Leiocolea rutheana Both L. rutheana var. rutheana and var. laxa are very rare plants of calcareous fens, a habitat which is very sensitive to disturbance, and both varieties are declining, having been lost from several historical sites. Culturing for cryopreservation and reintroduction programmes would be very prudent. Given var. laxa ’s status as a British endemic and its extreme rarity this should be a particular conservation priority.

Further research • Cryopreservation and reintroduction programmes to historic sites.

Fig. 8 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 15 samples of Leiocolea with 1 sample of Gymnomitrion concinnatum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. This analysis resolves L. rutheana var. rutheana and var. laxa separately and does not support their treatment as conspecific subspecies. Samples of L. rutheana s.s are resolved as a strongly supported clade showing some intraspecific variation, while var. laxa is resolved in a strongly supported clade with L. gillmanii .

41 4.2.10 Marsupella profunda Lindb. 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

This globally rare leafy liverwort species is restricted to disused and working china clay pits in Cornwall within Britain and is also known from Portugal and Macaronesia. It frequently grows in mixed stands with the more widespread species M. sprucei and the two species are very difficult to distinguish morphologically. Marsupella profunda . Photo: D.A. Callaghan Several sequences from related taxa generated in previous RBGE studies were available to augment this analysis. The DNA barcoding data were used to test whether M. profunda is genetically distinct from other morphologically similar British Marsupella species, particularly M. sprucei .

DNA Barcoding data 31 samples of Marsupella were used in the analysis, including 6 samples of M. profunda from Cornwall and Portugal and 7 samples of M. sprucei . Several misidentifications were flagged by the sequence data and most of these were resolved upon revisiting the voucher specimens. A combined analysis of data from all 4 barcoding regions (figure 9) resolved all samples of M. profunda in a well-supported clade distinct from all other species sampled. The data suggest that M. sprucei consists of two distinct lineages in Portugal and Britain.

Taxonomic implications For the target priority species M. profunda the DNA barcode data supports the effective species limits as currently recognised (e.g. Paton, 1999) and therefore no taxonomic changes are proposed. The analysis does however highlight additional complexity in M. sprucei which might represent cryptic species and requires further study. Frequent misidentifications were also flagged which suggests the morphological characters used to identify the species of this genus need to be revised.

Identification of Marsupella profunda The distribution and morphology of M. profunda mean that it is only likely to be mistaken for M. sprucei in the UK (with which it often grows intermixed). The two species can normally be distinguished by leaf characters, with M. sprucei having a shallower and broader sinus and more acute lobes than M. profunda . In cases of ambiguity either matK or ITS2 can differentiate M. profunda from the other species studied here.

Conservation status of Marsupella profunda Given the global rarity of this species it is of major conservation concern. In Britain it is restricted to disused china clay mines in Cornwall, and this habitat probably supports some of the best populations of the species across its known distribution. The populations in Cornwall appear to be relatively stable but continued monitoring is crucial.

Further research • Comparison with Macaronesian populations of M. profunda . • Further investigation into the variation shown between UK and European populations of M. sprucei .

Fig. 9 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 31 samples of Marsupella with 1 sample of Jungermannia atrovirens as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The tree shows M. profunda to be resolved in a strongly supported clade, reinforcing its treatment as a distinct species.

44 4.2.11 Orthotrichum pallens Bruch ex Brid. & O. pumilum Sw. ex anon. Taxonomic categories: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

Orthotrichum contains several groups of taxonomically critical species and the genus as a whole is often approached with trepidation by beginners. O. pallens & O. pumilum belong to one such variable and critical complex with several species recognised in Europe. Representatives of this complex in the UK are the 2 target Orthotrichum pallens . Photo: M. Lüth species, O. pallens & O. pumilum , as well as 2 other difficult small Orthotrichum species: O. tenellum and O. stramineum (Smith 2004). Some European authors also recognise O. schimperi as a species distinct from O. pumilum (Blockeel 2006), making this another important entity for comparison with British O. pumilum populations. This study aimed to use DNA barcoding data to assess species boundaries in the O. pumilum/O. pallens complex.

DNA Barcoding data In an analysis of combined data from all 4 DNA barcoding regions (figure 10) O. pallens was shown to be distinct from all other UK Orthotrichum species, although when compared to the morphologically similar Spanish species O. casasianum the barcode data failed to differentiate the two. Samples attributed to O. pumilum and O. schimperi from across Europe show considerable variation which is not consistent across the barcode regions sequenced. They group together in a larger clade containing 3-4 lineages, one of which has strong bootstrap support (93%) and contained all but one of the samples originally attributed to O. schimperi .

Taxonomic implications The data support O. pallens as a valid species, distinct from all other British species but not resolved from the Spanish endemic O. casasianum . This calls into question the status of this more recently described Spanish species, but that is beyond the scope of the present study. The genetic variation within the O. pumilum/schimperi group suggests that this group still require additional study on a broad scale. However, the strongly supported clade containing the majority of O. schimperi samples seems to support recognition of this

45 taxon. Some of the samples in this clade were originally identified as O. pumilum but on re-examination in light of the molecular data they were found to possess the morphological characters normally attributed to O. schimperi , while the other samples of O. pumilum appeared to be more variable. Both British samples which were available for sequencing fall within this clade and fit the morphological criteria of O. schimperi .

Identification of Orthotrichum pallens & O. pumilum/schimperi O. pallens is morphologically most similar to O. stramineum in the British flora but the combination of its small size, pale, orange-tipped hairless calyptra and hairless vaginula are diagnostic. O. pumilum is another very small species which resembles O. tenellum in many respects but with a short, hairless calyptra which only partially covers the capsule, unlike the long, slightly hairy calyptra of O. tenellum . Authors who recognise O. schimperi cite its smaller size and ventricose capsules with orange ribs as diagnostic (Blockeel 2006).

Conservation status of Orthotrichum pallens & O. pumilum/schimperi While the similarity of these species to more common relatives, coupled with the fact that many bryologists are unfamiliar with their subtle distinguishing features, might mean that they have been overlooked in the past, the increase in recent records of these and other epiphyte species is more likely a result of climate change and a decrease in levels of atmospheric pollution. Casual epiphyte colonisation events from the continent appear to be increasing (Bosanquet 2012) and it may be that this is the situation with these Orthotrichum species, in which case the native status of the species (or at least the establishing populations) is in doubt, with subsequent implications for their conservation status.

Further research • Additional study of the O. pumilum/O. schimperi complex is required. • The status of O. casasianum in relation to O. pallens warrants further investigation.

Fig. 10 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 41 samples of Orthotrichum with 1 sample of Bryum algovicum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves O. pallens in a moderately supported clade with the Spanish endemic O. casasianum . This supports the treatment of O. pallens as a distinct species in the British flora but undermines the status of O. casasianum as a distinct species. All samples of O. pumilum & O. schimperi are resolved in a single clade with good support but the additional structure within this clade suggests the group requires additional study.

47 4.2.12 Pallavicinia lyellii (Hook.) Carruth. 2) Species which may consist of 2 or more distinct cryptic species.

Pallavicinia lyellii is a simple thalloid liverwort and the only representative of the family Pallaviciniaceae in Europe. The species is easily distinguished from other liverworts when fertile thalli are present although these can sometimes be difficult to find. Indeed the plants themselves can be difficult to find in one of the habitats the species occupies- growing amongst Male plants of Pallavicinia lyellii . Photo: D. Bell hummocks of Molinia in boggy ground. P. lyellii also occupies a markedly different habitat in SE England where it forms large sheets on exposed sandstone in woodland. This extreme difference in habitats raises the question of whether these two ecologically diverse entities in fact represent two distinct species.

DNA Barcoding data Six out of the seven samples with DNA extracted were successfully sequenced for all four barcoding regions. The barcode data presented here do not support the presence of 2 cryptic species within P. lyellii in Britain, as all samples had identical sequences across all four barcoding regions, with the exception of a single substitution in ITS2 for one sample. It would however be interesting to study this group using additional molecular markers as globally the genus Pallavicinia is known to show very little genetic diversity (Laura Forrest, pers. comm.)

Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Paton 1999) and therefore no taxonomic changes are proposed.

Identification of Pallavicinia lyellii When fertile material is present P. lyellii is very distinctive with male plants having a set of fringed scales along each side of the midrib and female plants with a fringed involucre surrounding the perianth. The dark green thallus of sterile plants has very thin translucent margins which a markedly thickened midrib.

Conservation status of Pallavicinia lyellii Lockhart et al. (2012) note a general decline of this species across Europe. In Britain the populations on sandstone in southeast England appear to be healthy and stable, although they may be at some risk from climbers using the outcrops at Harrison’s Rocks in East Sussex. The bog populations in the north and west are at greater risk from habitat degradation and developments such as wind farms.

48 4.2.13 Rhytidiadelphus subpinnatus (Lindb.) T.J.Kop. 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species.

R. subpinnatus is a rare moss of woodlands with a scattered western distribution in Britain. It is morphologically intermediate between Loeskeobryum brevirostre and the ubiquitous R. squarrosus . This study used DNA barcoding markers to test whether R. subpinnatus is genetically distinct from morphologically similar species in Britain.

DNA Barcoding data Of 35 available samples all were sequenced successfully for rbcL , 24 for matK , 33 for trnH- psbA and 34 for ITS2. All four barcoding regions distinguished R. subpinnatus from all the related taxa included in the analysis (figure 11), although the three chloroplast regions each only separated it from R. squarrosus by a single base substitution, while ITS2 distinguished these species by 3 substitutions. This close relationship is Rhytidiadelphus subpinnatus . Photo: D.A. Callaghan illustrated in the strict consensus maximum parsimony tree of data from all four barcoding regions where R. subpinnatus is not resolved as reciprocally monophyletic in relation to R. squarrosus . Nevertheless the variation between the two species is consistent and supports the recognition of two distinct species.

Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Smith 2004) and therefore no taxonomic changes are proposed. Due to their close relationship treatment as a subspecies of R. subpinnatus could be argued but this might undermine its conservation value and since there are consistent morphological and molecular characters and no guidelines exist for determining rank based on genetic distances a conservative approach is recommended.

Identification of Rhytidiadelphus subpinnatus Intermediate in appearance between R. squarrosus and Loeskeobryum brevirostre , R. subpinnatus differs from the former in having shorter stem leaves which do not clasp the stem and from the latter by the absence of paraphyllia on the stem.

Conservation status of Rhytidiadelphus subpinnatus Although it appears to have been lost from many of the historical sites in England, the species has been reported as new to Scotland and discovered at several new sites in Wales in recent years. Bosanquet & Motley (2009) suggest it may be being overlooked elsewhere in Britain and note that the main threats to existing population are overgrazing by livestock and forestry activity.

49 Further research • Development of a standard protocol for using levels of genetic variation to assist in assessing taxonomic rank would be useful in cases such as this.

Fig. 11 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 35 samples of Rhytidiadelphus and relatives, with 1 sample of Eurhynchiastrum pulchellum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves R. subpinnatus in a strongly supported clade with R. squarrosus , which is further resolved with good support within that clade. Although they are not reciprocally monophyletic direct comparison of the sequence data shows that they are distinguishable genetically, with 6 substitutions across the four DNA barcoding regions.

50 4.2.14 Riccia bifurca Hoffm. & Riccia canaliculata Hoffm. Taxonomic categories: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

Riccia is a widespread genus of small complex thalloid liverworts which are taxonomically critical and difficult to identify. 13 species are recognised in Britain (Paton 1999). Many of these species are ephemeral, either on reservoir/lake margins or arable land. R. bifurca is a persistent terrestrial species which in Britain is restricted to the Lizard in Riccia bifurca . Photo: D.A. Callaghan Cornwall. It is very similar morphologically to several other persistent terrestrial and arable ephemeral species and identification is particularly difficult. R. canaliculata has long slender thalli and can be difficult to distinguish from terrestrial forms of the two aquatic Riccia species R. fluitans and R. rhenana . Several sequences generated in previous RBGE studies were available to augment the Riccia canaliculata . Photo: D.G. Long analysis. This study aimed to assess genetic diversity within this difficult genus in order to test the recognised species boundaries.

DNA Barcoding data 63 samples of Riccia were included in the analysis, covering every species known in Britain. Sequencing was generally successful for rbcL & matK but very poor for trnH- psbA & ITS2. Like Cephaloziella , many species of Riccia contain fungal symbionts, interference from which probably contributed to the low sequencing success rate in ITS2. Low primer specificity may account for the failed amplification in trnH-psbA . Analysis of the combined rbcL & matK datasets (figure 12) resolved several species including R. canaliculata with strong support. Samples of R. bifurca from Cornwall and the Netherlands were not resolved in the combined tree from R. subbifurca , R. crozalsii and two samples of R. beyrichiana from Cornwall (the remaining R.

51 beyrichiana samples from further north are resolved in a well-supported clade), while samples identified as R. bifurca from Germany and Sweden group with a sample identified as R. glauca from Latvia. However, direct comparison of the matK sequence data shows 9 base substitutions in matK differentiating the two UK R. bifurca and two R. beyrichiana samples from the rest of this group; the samples from the Germany/Sweden/Latvia clade all failed to amplify for matK . Several misidentifications which were highlighted by the sequencing results were resolved when the specimens were re-checked, but others could not be resolved satisfactorily in the timeframe (two samples of R. beyrichiana from Cornwall and R. glauca from Latvia).

Taxonomic implications The data support the current status of R. canaliculata as a distinct and conserved species. R. bifurca requires further study, particularly in relation to R. subbifurca , R. crozalsii and R. beyrichiana . Both samples of R. beyrichiana from Cornwall were growing in close proximity to R. bifurca and cross contamination from a mixed gathering is possible, but this seems unlikely to have happened twice and a re-assessment of the contents of the herbarium packets was inconclusive. The British R. bifurca samples also need to be studied further in comparison to the German and Swedish R. bifurca samples (and the Latvian R. glauca sample, which appears to be a misidentification). Additional attempts to obtain matK data for these samples would be desirable to determine the extent of the genetic variation between these populations as they may represent two distinct species. Unfortunately herbarium study of Riccia spp. is generally hampered by the tendency for the plants to shrivel upon drying and not regain their original form when rehydrated.

Identification of Riccia bifurca & R. canaliculata The long narrow thalli of R. canaliculata will distinguish it from all other species except terrestrial forms of R. fluitans and R. rhenana , both of which have more translucent thalli with readily visible areolae and lack a central groove. R. bifurca can be distinguished from R. crozalsii and R. subbifurca by the long- persistent orange-brown older regions of the thallus. R. crozalsii also differs in having numerous conspicuous cilia. R. beyrichiana can be distinguished from R. bifurca by spore characters when sporophytes are present, and is otherwise normally distinguished by the larger, more robust thalli with swollen margins.

Conservation status of Riccia bifurca & R. canaliculata R. canaliculata is an ephemeral species of reservoir and pond margins which requires prolonged periods with low water levels and exposed mud and is therefore at risk from changing water management regimes. Several historical sites for the species in Scotland were searched unsuccessfully during the course of this project, some of which now maintain high water levels throughout the year for fishing. The species may be declining generally in Britain with recent records only from sites in Shropshire and Midlothian. R. bifurca is a perennial species which in Britain is restricted to the Lizard in Cornwall and all populations are on protected land. The British material may represent a distinct taxon from European R. bifurca which would have implications for its distribution and conservation status.

52 Further research • Culturing (from spores where available) to reduce levels of contamination from fungal symbionts and other organisms. • Design more suitable primers for trnH-psbA . • R. beyrichiana from Cornwall requires closer investigation as it appears to differ genetically from other UK populations. • R. bifurca populations in the UK and Europe require careful comparison; more matK data would be useful to this end.

Fig. 12 Maximum Parsimony strict consensus tree for the combined dataset of rbcL & matK for 65 samples of Riccia with 1 sample of Conocephalum japonicum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves R. canaliculata in a distinct clade with strong support. R. bifurca is unresolved, but see text for discussion of direct sequence comparisons.

53 4.2.15 Sphaerocarpos texanus Austin 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 2) Species which may consist of 2 or more distinct cryptic species.

Sphaerocarpos is a distinctive liverwort genus represented by two species in Britain, S. texanus and S. michelii . Both species are rare southern plants and can only be distinguished from each other by subtle characters of spore ornamentation and therefore cannot be identified in the field. The DNA barcoding data were used to assess levels of genetic variation to test the validity of these species and search for additional intraspecific variation. Sphaerocarpos texanus . Photo: D.G. Long

DNA Barcoding data Previous work undertaken at RBGE by Daniella Schill on a global revision of this group, although as yet incomplete, meant that there were plenty of samples with DNA already extracted available for this study. matK and ITS2 both performed very poorly in terms of sequencing success, but both rbcL & trnH-psbA showed sufficient variation to distinguish the two British species, with an analysis of their combined datasets producing a well-resolved tree (figure 13). The analysis supports the hypothesis of cryptic species within both of the recognised taxa, with S. texanus containing two phylogenetically disparate lineages (European and North American), and S. michelii made up of several lineages, two of which are present in Britain.

Taxonomic implications The molecular data supports the current treatment in Britain of S. texanus as a species distinct from S. michelii , although it also shows that cryptic species exist within S. texanus , so a different name should be applied to the European plants. The appropriate name would appear to be S. europaeus , used by Lorbeer (1934) who treated European populations of S. texanus as a separate species based on cytological evidence. This treatment has not been followed by subsequent workers, but the molecular data presented here support Lorbeer’s treatment. Daniela Schill (pers. comm.) also identified subtle characters of the spores to distinguish the North American and European plants when working on the group at RBGE. Another interesting result from the barcode data which will have implications for the British bryophyte flora is the variation within S. michelii in Europe, with 4-5 lineages identified, two of which are present in the UK. The lineage from the Isles of Scilly in Cornwall appears to be unique while plants from Norfolk fall within the main European clade. Further study of this group is necessary to determine the full extent of molecular and morphological variation and the distribution of each lineage, and to determine at what rank these taxa should be recognised. Currently there is some evidence that spore size and subtle characters of the spore ornamentation can be used to distinguish between lineages (Daniela Schill, pers. comm.). However, as S. michelii was not listed as a priority species this is outside the remit of the current study.

54 Identification of Sphaerocarpos texanus (= S. europaeus ) The spore characters described in Paton (1999) are the most reliable morphological characters for identification of S. texanus : spore tetrads with 4-6 alveolae across the distal surface of each spore (6-8 in S. michelii ), alveolae 16-30µm wide (10-18µm in S. michelii ) and lamellae papillose, without spines (smooth with spines at the angles in S. michelii ).

Conservation status of Sphaerocarpos texanus (= S. europaeus ) Recognition of S. europaeus considerably reduces the distributional ranges of both species previously treated as S. texanus and this should be reflected in their conservation status.

Further research • Additional study of S. michelii is needed to determine the full extent of variation and find supporting morphological characters. • Morphological characters differentiating North American and European populations attributed to S. texanus need to be defined and the application of a name to the European taxon requires consideration.

Fig. 13 Maximum Parsimony strict consensus tree for the combined dataset of rbcL & trnH-psbA for 43 samples of Sphaerocarpos with 1 sample of Blasia pusilla as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves S. texanus in two unrelated clades, corresponding to North American and European samples and indicating that the ‘ texanus ’ epithet has been wrongly applied to European material. The analysis also reveals a large amount of genetic variation within S. michelii , suggesting that this species requires additional study.

56 4.2.16 Sphagnum balticum (Russow) C.E.O.Jensen 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 2) Species which may consist of 2 or more distinct cryptic species; 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

The widespread genus Sphagnum is one of the most ecologically important plant groups in the world but it contains many taxonomically difficult species. One such species is Sphagnum balticum , which grows as scattered stems amongst hummocks of other Sphagnum species in a handful of sites across Wales, northern England and Scotland. Sphagnum balticum . Photo: D.G. Long The DNA barcoding data were used to test whether S. balticum is genetically distinct from other morphologically similar species, particularly S. fallax .

DNA Barcoding data DNA extraction and sequencing of the Sphagnum samples was very successful for all 4 barcoding regions. In a combined analysis of the 4 regions (figure 14) the barcode data resolved S. balticum as a distinct and uniform taxon with a sister species relationship to S. fallax , with the exception of one sample originally identified as S. balticum which the barcode data flagged as a misidentification of S. cuspidatum . Other species with multiple accessions were also resolved with good support in the analysis.

Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Smith 2004) and therefore no taxonomic changes are proposed. The straightforward sequencing and excellent resolving power of the barcode data in this group shows great potential for application of the technique in this extremely ecologically important genus.

Identification of Sphagnum balticum Normally growing as scattered stems amongst other Sphagnum hummocks, S. balticum can usually be distinguished by the untidy appearance caused by the upper spreading branches rising above the centre of the capitulum, single juvenile branches visible between the rays of the capitulum (rather than pairs), spreading branch leaves and normally single pendant branches which do not clothe and obscure the stem.

Conservation status of Sphagnum balticum This species is very rare and declining in Britain. It is sensitive to habitat degradation and sporophytes are unknown in the UK so it is unlikely to colonise new sites. Protection and monitoring of existing populations is crucial and efforts should be made to culture material for cryopreservation and reintroduction programmes.

Further research • Cryopreservation and reintroduction programmes to historic sites.

Fig. 14 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 24 samples of Sphagnum with 1 sample of Oligotrichum hercynicum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves S. balticum as a distinct and uniform species with good support and shows no intraspecific variation.

59 4.2.17 Thamnobryum angustifolium (Holt) Nieuwl. & T. cataractarum N.G.Hodgetts & Blockeel 3) Species which are taxonomically problematic due to being in a genus where there is real uncertainty over species limits, with the problem involving many species in the genus.

In Britain Thamnobryum is represented by 4 species: the ubiquitous T. alopecurum , the presumed “garden escape” T. madarense , T. angustifolium and T. cataractarum . The last 2 species are endemic to Britain, known from just 2 sites and 1 site respectively. This study used DNA barcoding markers to test the validity of these species by assessing levels of genetic variation between them.

DNA Barcoding data Although matK data wasn’t available, sequencing of the other 3 barcoding regions was very successful. However, genetic variation was minimal with just 4 variable bases across all 3 barcoding regions and this variation was not consistent with morphological determinations. A maximum parsimony analysis of the data produced a poorly resolved and weakly supported strict consensus tree (figure 15).

Taxonomic implications These results do not support the concept of 4 distinct species of Thamnobryum in Britain. It is likely that the morphological variation observed is the result of physiological adaptations to the extreme riparian habitats the samples were occupying. This agrees with the findings of Olsson et al. (2009), although their results showed some structure based on the geography of samples. That study only included 2 British specimens (1 sample each of T. cataractarum and T. angustifolium ) which were resolved from a German sample of T. alopecurum and it may be that additional sampling of European samples would reveal some structure to the little variation found in the British samples used here.

Identification of Thamnobryum angustifolium & T. cataractarum The morphological characters used to differentiate these entities (leaf and costa width; degree of toothing on the leaf margin) appear to be environmental adaptations or random independent mutations and therefore recognition of these species is not supported.

Conservation status of Thamnobryum angustifolium & T. cataractarum These results suggest that these 2 species represent extreme habitat forms of the widespread T. alopecurum and would therefore warrant no special conservation efforts.

Further research • The Macaronesian species T. fernandesii was not available for study but this should also be sequenced for comparison, along with a broader range of European T. alopecurum .

Fig. 15 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, trnH-psbA & ITS2 for 14 samples of Thamnobryum with 1 sample of Eurhynchiastrum pulchellum as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The tree shows no significant structure with only 2 weakly supported clades which don’t correspond to the rare English species discussed in Smith (2004).

61 4.2.18 Tortula freibergii Dixon & Loeske Taxonomic categories: 1) Species which are taxonomically problematic due to uncertainty over their distinctness from one (or a few) more common congeneric species; 2) Species which may consist of 2 or more distinct cryptic species.

Tortula freibergii is a rare moss species of Western Europe with a disjunct distribution in Britain, having populations in Sussex, Yorkshire, Fife and an extended population along canal margins in Staffordshire, Shropshire, Cheshire and South Lancashire. It is closely related to T. solmsii and T. marginata , and was at Tortula freibergii . Photo: D.A. Callaghan one time treated as a subspecies of T. cuneifolia . Differentiating between these four species can be difficult and molecular data is desirable to test the species limits. T. freibergii also exhibits significant morphological variation between populations and this has led to speculation that different populations may represent distinct taxa. The DNA barcoding data were used to test these hypotheses.

DNA Barcoding data A total of 18 Tortula accessions were used in the final analysis, including six T. freibergii , three T. solmsii , three T. marginata , two T. cuneifolia and two samples each of T. truncata and T. subulata . Two additional accessions of T. solmsii had DNA extracted but were excluded from the analysis due to contamination. All samples were successfully sequenced for rbcL and only one sample (of T. truncata ) failed to amplify for trnH-psbA . Sequencing success was low for matK with both samples of T. cuneifolia and T. subulata , and one sample each of T. freibergii and T. solmsii failing to amplify, while only one partial matK sequence was obtained for T. marginata . Sequencing of ITS2 also failed for two samples of T. solmsii and both samples of T. truncata and T. subulata . Nevertheless, an analysis of the combined available data resolved each species with moderate to strong bootstrap support (figure 16). ITS2 was the only barcoding region which failed to distinguish between T. freibergii and T. solmsii , despite the otherwise strong resolving power of this region. None of the studied molecular regions showed any variation within British T. freibergii (and only a single base substitution in each of trnH-psbA and ITS2 between these and the Italian accession sampled), suggesting that all populations represent a single species and that documented morphological variation is not genetically controlled but rather due to environmental factors.

62 Taxonomic implications The DNA barcode data supports the effective species limits as currently recognised (e.g. Smith 2004) and therefore no taxonomic changes are proposed. A detailed morphological study carried out by University of Edinburgh student Catherine Kwella supports the conclusions of the molecular study, namely that populations of T. freibergii represent a single distinct species showing morphological plasticity and a disjunct distribution in Britain. The disjunct pattern of distribution could be explained by the specific habitat requirements of the species (exposed sandstone in shady coastal situations) and human influences, e.g. the relatively extensive population along canal margins in northwest England (Callaghan 2008).

Identification of Tortula freibergii The smaller spores, longer marginal cells and ability to develop papillae on the leaf cells distinguish T. freibergii from T. cuneifolia . The leaves of T. solmsii are consistently densely papillose and the leaf border is narrower than that of T. freibergii , while capsules tend to be shorter and wider towards the mouth.

Conservation status of Tortula freibergii This is a very rare coastal species with a very disjunct distribution across its range. Populations appear to be stable and the recent discovery of two new sites in Fife has considerably increased its range in Britain. It appears to have very specific habitat requirements and targeted searches of shaded coastal sandstone outcrops may reveal additional populations.

Further research • Targeted surveys of suitable habitat along the coast between disjunct populations would be worthwhile.

Fig. 16 Maximum Parsimony strict consensus tree for the combined dataset of rbcL, matK, trnH-psbA & ITS2 for 18 samples of Tortula with 1 sample of Ulota phyllantha as an outgroup. Bootstrap values greater than 50% are shown above branches. Voucher details can be found in Appendix 1. The analysis resolves T. freibergii in a strongly supported clade with no internal variation, supporting its treatment as a single distinct species.

64 5. CONCLUSIONS

Table 4: Summary of the key findings for each UK priority species and the discriminatory power of each DNA barcoding region in relation to these. Resolved Resolved Resolved by trnH- Resolved Taxon by rbcL? by matK? psbA? by ITS2? Taxonomic implications Atrichum angustatum no yes (1) yes (1) yes (1) Current status upheld Barbilophozia kunzeana yes (4) yes (8) yes (1) yes (6) Current status upheld Bryum gemmiparum Further study cf. B. dichotomum no DD yes (0) yes (1) complex required Bryum knowltonii no DD yes (1) yes (1) Current status upheld Bryum salinum no DD no yes (1) Current status upheld Bryum warneum Current status upheld, although further no DD yes (1) yes (5) study of aberrant plants desirable Bryum calophyllum yes (1) DD yes (1) yes (2) Current status upheld Bryum marratii yes (1) DD yes (2) yes (4) Current status upheld Cephaloziella baumgartneri DD DD DD DD Data deficient Cephaloziella calyculata Full taxonomic revision of genus yes (2) DD DD DD required Cephaloziella integerrima Full taxonomic revision of genus yes (2) yes (20) yes (6) DD required Cephaloziella nicholsonii Full taxonomic revision of genus no no no DD required Ceratodon conicus Data appears to support the current yes (1) DD yes (3) DD treatment, but additional study desirable Ditrichum cornubicum yes (2) yes (7) yes (1) yes Current status upheld Ditrichum plumbicola no yes (6) yes (1) yes Current status upheld Ephemerum cohaerens DD DD DD DD Data deficient Eurhynchiastrum pulchellum Current status upheld- no intraspecific no DD no no variation found (in UK) Leiocolea rutheana var. 2 species present, with var. laxa more rutheana yes (4) yes (22) yes (9) yes (34) appropriately treated as a variety or Leiocolea rutheana var. laxa no DD no no synonym of L. gillmanii Marsupella profunda no yes (3) no yes (6) Current status upheld Orthotrichum pallens yes (1) yes (0) yes (1) yes (9) Current status upheld Orthotrichum pumilum/ Additional complexity revealed- schimperi complex no yes (0) yes (2) yes (0) possible cryptic species Pallavicinia lyellii Current status upheld- no intraspecific no no no no variation found Rhytidiadelphus subpinnatus yes (0) yes (1) yes (0) yes (0) Current status upheld Riccia bifurca Requires further study- possibly 2 no no DD DD cryptic species present Riccia canaliculata yes (7) yes (21) DD DD Current status upheld Sphaerocarpos texanus 2 species present, with UK material Y Y DD DD distinct from USA populations Sphagnum balticum no yes (0) yes (1) yes (1) Current status upheld Thamnobryum angustifolium no no no no Species not supported by data Thamnobryum cataractarum no no no no Species not supported by data Tortula freibergii yes (2) yes (2) no yes (4) Current status upheld Notes: Numbers in parentheses indicate how many substitutions are unique to the taxon. A zero indicates the species has no unique substitutions but is resolvable by a combination of characters. DD: data deficient

65 The results presented here demonstrate the power of DNA barcoding as a tool for resolving taxonomic issues, particularly in often difficult and character poor groups such as bryophytes, and the findings will be useful to help to prioritise the distribution of resources for conservation management programmes. The resolving power exhibited by these molecular markers is exceptional, with even the relatively conserved rbcL gene able to distinguish many of the species studied. The success of the technique in distinguishing species offers great hope for identifying suboptimal and aberrant samples which might not be identified on morphological grounds alone. These results also highlight where further research efforts should be focussed, both within the target priority species and also the related taxa.

6. DISSEMINATION OF RESULTS

Results have been presented to amateur and professional bryologists at the British Bryological Society AGM at the National Museum of Wales in Cardiff, September 2011, the 3rd International Symposium on Molecular Systematics of Bryophtes at the New York Botanical Garden, June 2012, and the Botanical Society of the British Isles Biological Recording conference in Edinburgh, September 2012.

This report will be made available online from the RBGE website and circulated to contributing bryologists and interested parties.

Preparation of a manuscript detailing key outcomes is being prepared for submission to the Journal of Bryology. Data generated for particular groups such as Bryum & Tortula will contribute to additional publications prepared in collaboration with other researchers.

All sequence data will be publicly available from the BOLD database (http://www.barcodinglife.com) & GenBank (http://www.ncbi.nlm.nih.gov/genbank) upon publication of results.

ACKNOWLEDGEMENTS

We would like to thank all of the regional bryologists who provided specimens for the study, particularly David Holyoak, Tom Blockeel, Des Callaghan, Sam Bosanquet, Gordon Rothero, Fred Rumsey, Peter Martin, Richard Lansdown and Thomas Hallingbäck, and staff at NMW, BM, MAUAM and LG for arranging the loan of specimens. Staff at Natural England and Scottish Natural Heritage are thanked for their assistance with licences and landowners. Michelle Hollingsworth, Laura Forrest and Ruth McGregor are thanked for assistance in the lab and for allowing us to use liverwort sequences generated by them. Alan Forrest is thanked for supplying matK primers and advice. Aliquots of DNA extracted by Nik Pedersen for previous studies on Bryum at the Natural History Museum, London were also kindly supplied by Stephen Russell for use in this study. We are also grateful to Daniella Schill for sharing data on Sphaerocarpos .

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