J. Squirrell

How much effort is required to isolate nuclear microsatellites from plants

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range‐wide comparisons (European vs. North American populations), the EST‐SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST‐SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST‐SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank–order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of FST were obtained for all marker types, but whilst absolute values for genomic and EST‐SSRs were very similar (FST = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP FST = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)

Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P(99)) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (H(exp)) of 0.294. At the population level, mean P(99) = 56%, mean A = 1.81, and mean H(exp) = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean F(IS) = 0.002). There was significant deviation from panmixia associated with population differentiation (mean F(ST) = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle F(ST) estimates, a pollen to seed flow ratio of 1.43 : 1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.

Taxonomic complexity and breeding system transitions: conservation genetics of the Epipactis leptochila complex (Orchidaceae)

The genus Epipactis contains a problematical complex of autogamous taxa among which species limits are difficult to define. Different authors have treated these plants in different ways, some recognizing the different taxa as distinct species, others considering them as minor intraspecific variants. These contrasting treatments have a direct impact on the conservation resources and status such plants command; ‘endemic orchid species’ are perceived as having high conservation value, ‘localized minor variants’ are not. We used allozyme and chloroplast restriction fragment length polymorphism (RFLP) and sequencing analyses to investigate patterns of population genetic structure underlying the taxonomic complexity in this group. Populations of E. dunensis, E. leptochila and E. muelleri were homozygous and uniform for all loci studied here. There were, however, fixed genetic differences among these taxa. Comparisons with published data from the putative progenitor species for the autogamous taxa (the widespread, allogamous E. helleborine) suggest iterative origins of autogamy, rather than the self‐pollinating taxa all being merely mutational variants of a single autogamous lineage.

Extending glacial refugia for a European tree: genetic markers show that Iberian populations of white elm are native relicts and not introductions

Conservation policies usually focus on in situ protection of native populations, a priority that requires accurate assessment of population status. Distinction between native and introduced status can be particularly difficult (and at the same time, is most important) for species whose natural habitat has become both rare and highly fragmented. Here, we address the status of the white elm (Ulmus laevis Pallas), a European riparian tree species whose populations have been fragmented by human activity and is protected wherever it is considered native. Small populations of this species are located in Iberia, where they are unprotected because they are considered introductions due to their rarity. However, Iberia and neighbouring regions in southwestern France have been shown to support discrete glacial refuge populations of many European trees, and the possibility remains that Iberian white elms are native relicts. We used chloroplast RFLPs and nuclear microsatellites to establish the relationship between populations in Iberia and the Central European core distribution. Bayesian approaches revealed significant spatial structure across populations. Those in Iberia and southwestern France shared alleles absent from Central Europe, and showed spatial population structure within Iberia common in recognized native taxa. Iberian populations show a demographic signature of ancient population bottlenecks, while those in Central European show a signature of recent population bottlenecks. These patterns are not consistent with historical introduction of white elm to Iberia, and instead strongly support native status, arguing for immediate implementation of conservation measures for white elm populations in Spain and contiguous areas of southern France.

Morphological, ecological and genetic evidence for distinguishing Anastrophyllum joergensenii Schiffn. and A. alpinum Steph. (Jungermanniopsida: Lophoziaceae)

Abstract The liverwort Anastrophyllum joergensenii Schiffn., reported from Norway, Scotland, Alaska and the Sino-Himalaya is shown to consist of two distinct species, A. alpinum Steph. (treated before as a synonym of A. joergensenii) in the Himalaya, western China, Alaska and Scotland, and A. joergensenii Schiffn. s. str. in Norway, Scotland and western China. The two species are distinguished on genetic characters, size, leaf and perianth characters, and appear to have different ecological preferences. Anastrophyllum alpinum, although the more widespread of the two in Scotland, is there known only as non-fertile plants, whereas in the Sino-Himalaya fertile populations and sporophytes are not infrequent; in contrast, the rarer A. joergensenii can produce perianths in Scotland, Norway and Yunnan but androecia and sporophytes are unknown. The differences between the two are detailed and the ecology and distribution outlined.