The Taxonomic Status of Gladiolus Illyricus (Iridaceae) in Britain

The Taxonomic Status of Gladiolus illyricus (Iridaceae) in Britain Aeron Buchanan Supervisor: Fred Rumsey, Natural History Museum, London A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science of Imperial College, London Abstract First noticed officially in Britain in 1855, Gladiolus illyricus (Koch) presents an interesting taxonomic and biogeographical challenge: whether or not this isolated northern population should be recognized as a separate sub-species. Fundamental conservation issues rest on the outcome. Here, the investigation into the relationship of the G. illyricus plants of the New Forest, Hampshire, to Gladiolus species across Europe, northern Africa and the middle east is initiated. Two chloroplast regions, one in trnL–trnF and the other across psbA–trnH have been sequenced for 42 specimens of G. illyricus, G. communis, G. italicus, G. atroviolaceus, G. triphyllos and G. anatolicus. Phylogenetic and biogeographical treatments support the notion of an east–west genetic gradation along the Mediterranean. Iberia particularly appears as a zone of high hybridization potential and the source of the New Forest population. Alignment with sequences obtained from GenBank give strong support to the classic taxonomy of Gladiolus being monophyletic in its sub-family, Ixioideae. Comments on these chloroplast regions for barcoding are also given. In conclusion, the genetic localization of Britain’s G. illyricus population as an extremity haplotype suggests that it could well deserve sub-species status. Contents <ul style="display: flex;"><li style="flex:1">1 Introduction </li><li style="flex:1">2</li></ul><ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">2 Background </li></ul>3 Materials and Methods 4 Results and Discussion 5 Conclusions 8 15 26 28 56 Appendices References 1. Introduction G. illyricus in Britain Figure 1: G. illyricus flower spike. Photograph: Fred Rumsey, Denny Wood, New Forest. 1 Introduction The study of Gladiolus illyricus (Koch) is particularly pertinent at this time. It is a medium-sized flowering monocotyledon with a Mediterranean distribution, but with one important exception: the small yet significant, and now declining population in the New Forest of Hampshire, here in the British Isles. The natural history of G. illyricus in the UK, until as recently as the 1950s, ranges from being unclear to unknown. The first recorded sighting was on the Isle of Wight in 1855, with notes of its presence in the New Forest appearing soon thereafter. The origins of this population, in the extreme north of the species’ range, immediately became a point of debate. Was the arrival part of a natural progression northwards or was it an artificial, human-mediated introduction? Today, the continuing survival of G. illyricus in the New Forest appears fragile, making the above an important question. It is currently a protected Schedule 8 species and so protected under law by the Wildlife and Countryside Act, 1981, but with its endemic status unresolved, it remains on the waiting list of Cheffings and Farrel (2005)∗. The waiting list is populated by “taxa for which questions still remain over taxonomic validity or endemic status.” Indeed, the example of such a taxon given by the authors is G. illyricus ssp britannicus. This state of conservation limbo is unsatisfactory, for if the endemic sta- ∗‘The Vascular Plant Red Data List for Great Britain’ is part of the Joint Nature Conservation Committee’s Species Status project, which is used to inform the statutory five-yearly review of Schedules 8 species. 21. Introduction G. illyricus in Britain tus is rejected then much demanded conservation resources can be redirected elsewhere. However, if it is confirmed, a fuller more concerted effort would be justified. The issue of the British nativity of G. illyricus is theoretically inseparable from the suggestion that the population in the New Forest should be designated as being a separate subspecies, G. illyricus ssp britannicus. This study was born with the desire to resolve the taxonomic status of G. illyricus and hence shed light on the natural history of the New Forest population. Resolution would thus make clear the level of impetus required of conservation efforts. An outline for a study to answer the question has been proposed by Lockton (2006); that is, to demonstrate: 1. the existence of an east–west genetic gradation 2. that the gradation is correlated to natural dispersal rates. 3. that there is speciation within that gradation. 4. that the British plants are sufficiently distinct. 5. that they belong to a distinctive semi-natural vegetation habitat. The ecological investigation, that can make clear the issues of the fifth point has already started, most notably by Stokes (1987). This study begins the molecular investigation of the G. illyricus population of the New Forest, and hence starts to address the issues of the first, third and fourth points. The work began with the important grounding step of placing the European Gladiolus species into a wider context. Gladiolus is one of the largest genera in the Iridaceae family, with possibly as many as 300 species. Almost all of these grow in sub-Saharan Africa (Goldblatt et al. 2001); less than twenty species are endemic to Europe and neighbouring countries. Support for the monophyletic status of all Gladiolus is needed to be able to rely on existing work that assumes the long standing taxonomy. After the broad initial analysis, the project then focussed on the inter-relations within Europe, which were then explored with the above framework in mind. Sadly, the full answer can not be given here: limited time restricted the scope of what was achievable. However, important insights into the wider situation are revealed, providing some answers and raising significant new ones, thus informing future research. 32. Background G. illyricus in Britain Figure 2: An example of G. illyricus from the herbarium at Reading University. This is a New Forest plant, collected in 1960. 2 Background The first systematic study of Gladiolus illyricus (Koch) growing in the UK did not start until almost one hundred years after it was first officially noticed. As such, our knowledge of its distribution and ecology is very sparse for times before 1950, when Bowman started the surveying of the New Forest for this purpose (Rand, 2005), and almost completely blank for anything before 1855, when Mrs Phillipps of the Isle of Wight made the first record of its growing in the British Isles (More, 1862). According to Townsend (1904) the first mainland discovery was in 1856 by Rev. W. H. Lucas. From the mid to the end of the 19th century, the story seems to be one of undirected specimen collection with the occasional serendipitous discovery of new sites. The Natural History Museum’s British Herbarium holds thirty nine specimens from this period (the first being the 1858 sheets of J. T. Boswell-Syme), all collected from within the New Forest (v.c.11), seldom with a more detailed description of location than “near Lyndhurst”. Accompanying notes are generally sparse as well, although Rand (2005) points out that descriptions of habitat, beyond “amongst bracken”, were sometimes made. The most detailed of these are by Dyer and Trimen (1864), but they worked on only two sites in the New Forest, within less than 3km of each other. Later, Townsend (1904) compiled a summary of the New Forest locations where it had been found, describing three almost distinct areas of modest size, all within 6km of Lyndhurst. Crucially, Pope et al. (2005) 42. Background G. illyricus in Britain notes that it was also found near Ensbury, Dorset (v.c.9) around 1874 and collected trice more from the Isle of Wight in 1872, 1897 and 1931, although the last collection was rather haphazard. The plants seems to have then received little attention from the turn of the century until the systematic surveys of Bowman from the 1950s onwards, Hamilton in the 1960s and Everett in the 1980s (Rand, 2005), as well as Stokes (1987). From their foundational reports and the continued efforts of the New Forest Study Group, we are now fairly certain of the full extent of the sub-populations growing in the New Forest. The widespread activity of botanists across Britain means that we can now be sure that G. illyricus no longer grows outside this area. The current range, while still small, is several times larger than that investigated by botanists at the turn of the century, spreading up to 12km from Lyndhurst. What the actual distribution was in those early years is almost impossible to deduce, but it seems reasonable to surmise, with sub-populations growing outside the New Forest at the time, that it was more consistently widespread then, than it is today. However, this is a pivotal historic factor for the question of G. illyricus being endemic in the New Forest: care must be taken before assumptions are made. Was the sudden start of botanical interest in the middle of the 1800s perhaps a result of a contemporary introduction to the south coast from a continental population? I believe not: it is most likely due to artifacts of historical circumstances and not because of any events or trends belonging to the plants themselves over the period in question. Indeed, Mrs Phillipps did not luckily stumble upon the singular occurrence of G. illyricus on the Isle of Wight, but rather that the “Wild Gladiolus” plant had been growing in the woods at Shanklin, the nearby town, for at least a generation (More, 1862). It is quite possible that, had local people written of the flowers that they commonly saw, or such notes more widely disseminated, records of many plants would have appeared much earlier. Martin Rand (2005) makes a good case for the existence of G. illyricus in the New Forest before the first records appeared. He points out that many species, clearly native in the New Forest, were not officially noticed as growing there for a very long time (Carex montana: 1876 and Gallium constrictum: 1924, for example). He adds to that a description of the difficulty of finding G. illyricus colonies, even when in flower. Another possibly relevant issue is that access to the New Forest became easier after the railway through it opened in 1847. Furthermore, Rand collects a variety of ecological observations to support the idea that G. illyricus has been in Britain for some time. Despite its restricted distribution, it appears in a range of ecotones with a variety of associations, none of which are guaranteed. Also, it seems to bare an interesting relationship with semi-natural habitats (some dating back thousands of years), to which it 52. Background G. illyricus in Britain is seemingly well adapted. Overall, a strong sense of long-term naturalization is given by what we do know, although the evidence frustratingly does little to dismiss the neophyte hypothesis outright. However, the notion of a very recent introduction can be disregarded: that there was a prolific dispersal of a plant sensitive enough to just about immediately fall into decline, or that there was some artificial, yet persistent effort to spread the flower across Hampshire are highly improbable. It seems certain, therefore, that it did not arrive just in time for the botanists getting off the first trains ever to arrive at Lyndhurst, but has it been here long enough to be considered native? The debate on the endemism of G. illyricus started alongside the first reports of its discovery. Alexander More published his opinion in the article revealing the 1855 specimen (More, 1862). He was convinced that it was indigenous to Britain: the remoteness of the Isle of Wight location, the lack of any known garden cultivations and the similarity of the growing environment between the plants in the UK with those undisputable natural populations in north western France, together strongly supported the notion of a natural progression along the Atlantic coast from the Mediterranean. As Toone notes in his review of this topic (2005), by the end of 1863 Borrer, Babington and Boswell-Syme had all agreed that the case for it being a native species was strong. Mansell-Pleydell (1874) added himself to this list in his ‘Flora of Dorset’. H. C. Watson provided the only opposition, suggesting the possibility of an association with planted trees or shrubs. Townsend (1904) reported the idea that it could have been introduced, along with other species, with the imports, around 1800, of young fir trees to Bournemouth from the Landes area of France (where a similar G. illyricus was known to grow). However, evidence does not support any correlation between the growing areas of these two species. As such, I think it is reasonable to assume that the first records were not an account of a spreading introduction, but rather the initial documentation of an established population. A picture emerges that suggests a lower bound on the age of the Britain plants is of the order of many hundreds of years. It could be considered even higher, depending on the rating of the likelihood of jump dispersal events. Assuming that the Isle of Wight population was once a part of a mainland range puts the lower bound at being over 7000 years ago (Cooper and Jay, 2002). Rejecting long range dispersal altogether and therefore assuming that G. illyricus arrived in Britain by migration, fixes the arrival time to before the last formation of the Channel, about 8000 years ago. The upper bound is a little more certain, being defined by the timetable for the increase in temperatures since the last glacial maximum, over which the ice sheet and permafrost retreated north allowing plants growing in the refugia along the Mediterranean to rapidly 62. Background G. illyricus in Britain spread north and recolonize Europe (Hewitt, 1999). Temperatures started to rise again from about at least 18,000 years ago (the lower bound for the last glacial maximum or LGM), at which point southern Britain, connected to France at the time, was within the limits of the permafrost and mostly covered by the ice sheet of northern Europe (Taberlet et al. 1998). After a period of warming, the Younger Dryas stadial (Berger, 1990) meant Britain still had sub-zero mean annual temperatures until about 11,500 years ago (Atkinson et al. 1987). This ended abruptly when temperatures rose by as much as 7◦C in a matter of decades and then steadily increased to near present averages (Alley, 2000). The climate of the south coast of England permanently reached temperatures suitable for G. illyricus maybe as soon as 1000 years later. Migration is an additional limiting factor: after the LGM, the maximum migration rates achieved were an impressive, but bounding, 2km per year (Bennett et al. 1986). It seems, therefore, that G. illyricus was probably not growing in the precursor of the British Isles much before 10,000 before present. How long after this G. illyricus could have actually arrived would be nothing more than guess at this point. Today, there is a definite trend of decline (Brewis et al. 1996). For example, a search in 1947 concluded that it gone extinct on the Isle of Wight since the last sighting in 1931. Within the New Forest itself, the smaller sub-populations are slowly disappearing. The matter might be all the more pressing for the fact that its decline is also being seen in France. Sub-populations are known to have gone extinct, including the crucial Belle Isle (Brittany) population, which is thought to be the most closely related continental population to the British plants (Stokes, 1999). Like across the whole of North West Europe, a huge percentage of France’s heathland has been lost over the last 200 years (mainly to agriculture) and this is undoubtedly a significant factor. A trip to France undertaken to collect samples for this project at known, albeit almost historic, locations (Abbayes et al.; Boreau, 1857; Coste, 1906; Lloyd, 1844; Lloyd, 1886; Souche´, 1901) found that in most places, areas of suitable habitat were nowhere to be seen. The most promising locations were on rocky, heathered slopes above the River Argenton between Argenton-Chaˆteau and Thouars, but no sign of any Gladiolus were found. This author would not be surprised to discover that G. illyricus is extinct in the Loire Valley. Hence the importance of determining the resources and effort that should be spent on the conservation of the species here in Britain now. 73. Materials and Methods G. illyricus in Britain number of specimens 12 successfully sequenced (total = 49) failed to sequence (total = 17) 543210year Figure 3: Success in the sequencing of the herbarium specimens, influencing which were selected for processing. Keeping track of sequencing successes meant that it could be safely guessed that the last six oldest samples were unlikely to succeed and effort was spared by not attempting extractions. 3 Materials and Methods Leaf samples were obtained from two sources. Firstly, a licensed field trip to the New Forest at the end of the fourth week in June provided 52 small leaf samples, each stored separately and immediately in silica filled bags. All New Forest specimens were identified as G. illyricus. Locations are listed in Appendix A. Secondly, samples of G. illyricus, G. communis, G. italicus, G. atroviolaceus, G. triphyllos, G. anatolicus, G. imbricatus and G. palustris, collected from around the Medi- terranean over the last fifty years, were taken from specimen sheets in the herbarium at the University of Reading. Full details are listed in Appendix B. Selection was mainly dictated by availability. Specimens collected more than fifty years ago were omitted due to the poor outlook on DNA sequencing success, although specimens that might potentially have become key to uncovering relationships were kept. Sheets that were overtly of poor quality or paucity were also overlooked. Sixty two sheets remained from which 0.5–3cm2 of leaf material was taken. Care was also taken to ensure that the original determination was plausible. Six specimens were considered incorrect and eleven were possibly incorrect (notes are given alongside the relevant entries in the appendices). Tutin et al. (1980) was mostly used for the keys, with the complication that only a subset of the characters could be used, because the 83. Materials and Methods G. illyricus in Britain plants were dried herbarium specimens. Many of the taxonomically informative distinctions between these species are in the morphology of the flowers, including the perianth. Those used for differentiation were as follows. Between G. illyricus and G. communis: plant height leaf length leaf width no. flowers G. illyricus <ul style="display: flex;"><li style="flex:1">25–50cm </li><li style="flex:1">10–40cm 4–10mm </li></ul>30–70cm 5–22mm 3–10 G. communis 50–100cm 10–20 i.e. whether the specimen was large or small. The distinction between G. communis ssp. communis and ssp. byzantinus was not made, because over the limited range and quality of specimens available, a consistent separation was not observed. The principal difference used for the differentiation of G. italicus from G. communis (both having a highly overlapping range of values for the above size characters) was the anther-filament length ratio: G. communis anther shorter than filament G. italicus anther longer than filament Ambiguity arose when they were the same length, although exact measurements were not taken because the of the fragility of the herbarium specimens and the invasiveness of such measuring. The one G. palustris specimen was very hard to distinguish from G. illyricus. Differences between the other species encountered were more definitive. Key characters used include: <ul style="display: flex;"><li style="flex:1">species </li><li style="flex:1">most distinct character </li><li style="flex:1">notes </li></ul>G. atroviolaceus flowers deep violet-purple well preserved on herb. sheets G. anatolicus G. imbricatus G. triphyllos seeds unwinged dense flower spike leaf width<5mm from note on herbarium sheet but few flowers noticeably “straggly” The last species, G. triphyllos, is endemic to Cyprus and so was keyed out using Meikle (1985). Primer Selection and Sequencing To ensure success of the field collecting, the New Forest trip had to wait until it was certain that any potentially flowering plants were actually in flower. The knock-on effect was to delay the laboratory work. As such, reliable markers were chosen for sequencing targets. Reliable in this context means high copy, monomorphic genome regions of which 93. Materials and Methods G. illyricus in Britain the laboratory had experience (there would have been no time for experimentation and optimizations). Three chloroplast regions were picked out as ideal targets: • trnL–trnF (Taberlet et al. 1991) 0050 50 (forward) “C”: (reverse) “F” : <CGA AAT CGG TAG ACG CTA CG>3 <ATT TGA ACT GGT GAC ACG AG>3 • trnC–trnD (Demesure et al. 1995; Shaw et al. 2005) 0050 (forward) ‘trnC’: <CCA GTT CAA ATC TGG GTG TC>3 50 (reverse) ‘ycf6’: <TAC CAT TAA AGC AGC CCA AG>3 • psbA–trnH (Sang et al. 1997; Shaw and Small, 2004) 050 (forward) ‘psbA’: <GTT ATG CAT GAA CGT AAT GCT C>3 50 0(reverse) ‘trnH’: <CGC GCA TGG TGG ATT CAC AAT CC>3 These are all universal plastid primers. The trnLF region encompass the trnL intron, the 30 trnL exon and the trnL–trnF intergenic spacer. For the Gladiolus species sequenced it was found to be 733–742 bases long. It is a popular region, but very unpredictable in its variability (Shaw et al. 2005), showing very low rates of change for some groups (e.g. Sang et al. 1997). In the trnCD region, the trnC–ycf6 non-coding region was selected as being likely to be of a good size for trouble-free sequencing (i.e. 500– 1000 base pairs long). This is a less used region and cross-referencing its use is made tricky by the fact that the ‘ycf6’ gene is also known as ‘petN.’ Lee and Wen (2003) have a reverse primer for the same region that they call petN2R which overlaps greatly with the one used here, but whose ends are shifted distally about six bases. The primers employed for this study isolate the trnC–ycf6 intergenic spacer and some of the ‘ycf6’ gene itself. Finally, the psbAtrnH region has been highlighted as a good region for barcoding angiosperms (Kress et al. 2005). It is considered the most variable region of the three plastid regions above. From the results of this study, northern hemisphere Gladiolus have a slightly higher than average number of bases in this region: 609–616. When results from the chloroplast primers were ensured, an ITS region was also investigated. Although, almost always polymorphic (chromosome heterogeneity and diverging paralogues), it often shows high intra species variation, making it an excellent marker for tracing relationships (Baldwin et al. 1995). Furthermore, because it is a nuclear gene region, information from would perfectly compliment that from the chloroplast sequences, because of the differences in inheritance.

The Taxonomic Status of Gladiolus Illyricus (Iridaceae) in Britain

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