Research article
A multi-faceted approach for assessing evolutionary significant conservation units in the endangered Omphalodes littoralis subsp. gallaecica (Boraginaceae)

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Abstract

Genetic diversity is now regarded as a key component of biodiversity and its assessment has become a frequent addition to conservation studies. However, due to practical limitations, most studies assess genetic variation using neutral markers while the variability of evolutionary relevant quantitative traits is typically overlooked. Here, we simultaneously assessed neutral and quantitative variation in an endangered plant to identify the mechanism behind their spatial arrangement and to propose conservation guidelines for maximizing mid- to long-term survival. Omphalodes littoralis subsp. gallaecica is a self-fertilizing therophyte with an extremely narrow and fragmented distribution. Regardless of the marker set (non-coding sequences of cpDNA or Amplified Fragment Length Polymorphism loci), the five extant populations of O. littoralis subsp. gallaecica showed minimal to none neutral genetic diversity and a lack of gene flow between them. Moreover, genetic structure was identical in samples collected on two consecutive years suggesting that the seed bank cannot buffer against genetic loss. High rates of self-fertilization together with a strongly fragmented distribution and recurrent bottlenecks seem the likely mechanisms that may have led to a dramatic loss of genetic variation in a classic scenario drawn by genetic drift. Despite the extremely narrow distribution range, reciprocal transplant experiments revealed that the populations differed in several quantitative traits and that these differences likely have a genetic basis. Nevertheless, the pattern of differences among populations did not fit the expectations of local adaptation. Instead, phenotypic variation seemed another outcome of genetic drift with important implications for conservation because each population should be designated as an independent evolutionary significant unit (ESU). Our study evidences the benefits of combining neutral markers with appropriate assessments of phenotype variation, and shows that even endemics with extremely narrow ranges can contain multiple conservation units.

Introduction

Designing and implementing appropriate measure that enhance the long-term survival of populations is a major challenge in conservation (Ellstrand and Elam, 1993). In this regard, the genetic structure of endangered populations has become a primary focus of research since theory predicts that intraspecific genetic variation is pivotal for the persistence of species (Ouborg et al., 2006). Under the premise that populations may achieve their greatest evolutionary potential by maximizing their genetic diversity, conservation efforts often aim to preserve the most divergent populations and/or those displaying the largest levels of variation (Moritz, 1994).

Due to practical limitations, the genetic structure is usually assessed with neutral molecular markers even if their suitability for conservation purposes has been repeatedly questioned (Landguth and Balkenhol, 2012, Reed and Frankham, 2001). Instead, quantitative traits are those of most concern for conservation because they are directly related to the species’ fitness (Frankham et al., 2010). As natural selection act directly on phenotypes, not on genotypes, these traits reflect the species’ ability to undergo adaptive evolution as well as the consequences of inbreeding and outbreeding on reproductive fitness (Allendorf and Luikart, 2012). Unfortunately, current evidence suggests that neutral variation may not be an accurate indicator of quantitative variation; consequently, making decisions based only on genetic differences detected by neutral markers is not without risk (Frankham et al., 2010, Hedrick, 2001, Landguth and Balkenhol, 2012, Reed and Frankham, 2003). Nevertheless, knowledge of neutral diversity levels is useful for determining genetic relationships among individuals, among populations (gene flow and population structure), or the demographic history of the species (Reed and Frankham, 2001). In this context, a multifaceted approach that combines neutral and phenotypic data should provide a more comprehensive picture of the genetic structure, eventually leading to better conservation management.

Phenotypic variation among individuals results from the interaction between genotype and environment (Kawecki and Ebert, 2004). In the absence of other forces, populations are expected to develop traits that provide an advantage under their local environment resulting in a pattern where resident genotypes are better fitted to their local conditions than genotypes from other habitats (Williams, 1996). This pattern is known as local adaptation (Ashton and Mitchell, 1989). Nevertheless, local adaptation may be hindered by gene flow, confounded by genetic drift, and constrained by a lack of genetic variation (Lenormand, 2002). Disentangling whether the variation observed in phenotypes results from genetic differences, from environmental influence or from both forces acting additively is challenging because genotypes cannot be directly inferred from observed phenotypes (Frankham et al., 2010). Instead, reciprocal transplants are required to evaluate the relative contribution of phenotypic plasticity and genetics (Kawecki and Ebert, 2004).

From a conservation perspective, rare and/or endemic plants are of great concern because of their intrinsic characteristics: small population size, habitat specificity, and geographic isolation (Frankham et al., 2010). These features can be detrimental for the evolutionary potential of the species due to low genetic diversity, strong genetic drift, and inbreeding depression (Cole, 2003, Frankham et al., 2010, Höglund, 2009, Willi et al., 2006). However, rarity is only one of several factors known to have an impact on the species’ genetic structure. Life history traits, particularly life form and breeding system, have long been recognized as greatly influencing the distribution pattern of genetic diversity in plant populations (Hamrick and Godt, 1996). Namely, selfing species can maintain lower levels of genetic diversity and higher levels of differentiation among populations compared to outcrossers (Hamrick and Godt, 1996, Nybom, 2004).

The small annual Omphalodes littoralis subsp. gallaecica (Boraginaceae) M. Laínz (1971) is a rare herb (total occupancy <100,000 m2) restricted to coastal dune systems in northwest Iberian Peninsula (Romero Buján, 2005, Serrano and Carbajal, 2011). In the last decades, its populations experience continuous decline due to the threats faced by its sensitive habitat (Bañares et al., 2004); as a result, its current distribution is extremely fragmented and today the plant is known to occur in just five dune systems. Because of this rarity, O. littoralis subsp. gallaecica is cataloged as “endangered” by both the IUCN and the Spanish Catalogue of Threatened Species (Serrano and Carbajal, 2011, Ministerio de Medio Ambiente y Medio Rural y Marino, 2011), and listed as a priority species in the EU Habitats Directive (92/43/EEC, Annex II). Additionally, its habitat is considered as a Site of Community Importance (SCI) within the Natura 2000 network. O. littoralis subsp. gallaecica is a non-clonal, self-compatible plant and autogamy has been suggested as the most probable mechanism of reproduction (Bañares et al., 2004). Nevertheless, insects are often attracted to its flowers suggesting that in some cases it might reproduce by outcrossing (R. Retuerto, pers. comm.). Flowering period is very short, from March to April, and the ephemeral flowers last less than three days (Romero Buján, 2005). Each fruit develops four seeds that are mainly dispersed by exozoochory (Bañares et al., 2004). The seeds have specialized hooks that easily attach to the animal's fur (Bañares et al., 2004). Seed germination is high (R. Retuerto, pers. comm.) suggesting a small, viable seed bank. Population fluctuate greatly between years, multiplying or dividing by up to ten the number of their individuals (Bañares et al., 2004). The total number of individuals has been reported to be below 140,000 for the species with estimates per site ranging from 740 (our XN site) to 43,000 (our PC and DN sites) (Serrano and Carbajal, 2006). However, these estimates were published as part of the information provided in the IUCN Red List with no further detail on how they were obtained.

Despite the status of O. littoralis subsp. gallaecica as a species of conservation concern, its population genetics and the variation of its ecophysiological traits between populations have, to our knowledge, never been addressed. Here, we aim to fill this gap with an exhaustive molecular and phenotypic study of the five extant populations of this rare herb. We used sequences of the chloroplast DNA trnT-F region and genotypes derived from mostly-nuclear Amplified Fragment Length Polymorphism (AFLP) markers to address the following questions: (a) Is O. littoralis subsp. gallaecica genetically impoverished as it might be suggested by its life history traits? (b) Are its populations significantly differentiated from each other? (c) Given that O. littoralis subsp. gallaecica is a therophyte, are there significant between-year differences in its genetic structure? On the other hand, we performed a series of reciprocal transplant experiments to investigate the adaptive component of several quantitative traits related to fitness. Phenotypic variation was examined with an aim to answer: (d) Are there any phenotypic differences between populations? if so, (e) do these differences result from phenotypic plasticity or do they have a genetic basis? (f) Are they adaptive? Finally, we combined the molecular and phenotypic information to propose specific guidelines for the conservation of this endangered plant.

Section snippets

Leaf sample collection and DNA extraction

Samples for genetic analyses were collected on two consecutive years (2009 and 2010). In March 2009, plants (31–34 per site) were randomly sampled along the whole area occupied by the species at each of the five dune systems currently inhabited by O. littoralis subsp. gallaecica (Fig. 1). Minimum and maximum distances by the coast between study sites were 21 km (PC-TC) and 153 km (DN-XN), respectively. In four out of the five locations (DN, BD, TC and XN), plants occurred in dune slacks behind

Genetic diversity and structure

A total of 276 reproducible AFLP markers were scored in the 165 individuals sampled in 2009. Eighty-one (29.35%) loci were segregating for the whole data set and were retained for population's diversity estimates. Overall, 26 private bands were detected in all populations: one in population DN; two in BD, PC, and TC each; and 19 in XN (Table 1). Estimates of total genetic diversity for the species (He = 0.356; HSW = 0.530) were one or two orders of magnitude higher than the values observed at

Discussion

Taxa listed as endangered by the IUCN Red List of Threatened Species are considered to face a very high risk of extinction in the wild (IUCN, 2012). In the particular case of O. littoralis subsp. gallaecica, its status as endangered was granted attending to criteria of area of occupancy only: the plant occupies 10 hectares (well below the threshold of 500 km2 used by IUCN for endangered species), this area of occupancy is in continuing decline due to many threats, and populations sizes fluctuate

Acknowledgements

We would like to thank two anonymous referees for their helpful comments on an earlier version of the manuscript. Research supported by grant 07MDS031103PR and grant 00MDS006200PR (Xunta de Galicia). L. L. acknowledges support from Universidade da Coruña (contratos predoutorais UDC 2012).

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