The Use of Dna Barcoding to Address Major Taxonomic Problems for Rare British Bryophytes
Total Page：16
File Type：pdf, Size：1020Kb
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