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A combination of molecular markers (PCR-RFLP, AFLP) identifies

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A combination of molecular markers (PCR-RFLP, AFLP) identifies

    1 Targeting genetic resource conservation in widespread species: 2 a case study of Cedrela odorata L.

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    12134 Cavers S, Navarro C and Lowe AJ.

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    17 Centre for Ecology and Hydrology-Edinburgh, Bush Estate, Penicuik, 8 Midlothian EH26 0QB, Scotland, UK.

    29 Centro Agrónomico Tropical de Investigación y Enseñanza, Cartago, Turrialba 10 7170, Costa Rica.

    3 11 School of Life Sciences, University of Queensland, St Lucia, Brisbane, QLD

     12 4072, Australia

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    14 Address for correspondence: Stephen Cavers*, Centre for Ecology and 15 Hydrology, Edinburgh, Bush Estate, Penicuik, Midlothian EH26 0QB, Scotland,

    UK. Tel. 0131 445 4343, Fax. 0131 445 3943, *Email scav@ceh.ac.uk 16

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    18 Running head: Conservation of genetic resources in Spanish Cedar

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1 Abstract

    2 Conservation of genetic resources is a recognised necessity for the long term 3 maintenance of evolutionary potential. Effective assessment and implementation 4 strategies are required to permit rapid evaluation and protection of resources. 5 Here we use information from the chloroplast, total genome and quantitative 6 characters assayed across wide-ranging populations to assess genetic resources in 7 a Neotropical tree, Cedrela odorata. A major differentiation identified for

    8 organelle, total genomic and quantitative variation was found to coincide with an 9 environmental gradient across Costa Rica. However, a major evolutionary 10 divergence between the Yucatan region and Honduras/Nicaragua identified 11 within the chloroplast genome was not differentiated using quantitative 12 characters. Based on these and other results, a three-tiered conservation genetic 13 prioritisation process is recommended. In order of importance, and where 14 information is available, conservation units should be defined using quantitative 15 (expressed genes), nuclear (genetic connectivity) and organellar (evolutionary)

    measures. Where possible, information from range wide and local scale studies 16

    17 should be combined and emphasis should be placed on coincidental disjunctions 18 for two or more measures. However if only rapid assessments of diversity are 19 possible, then assessment of organelle variation provides the most cautious 20 assessment of genetic resources, at least for C. odorata, and can be used to

    21 propose initial conservation units. When considering effective implementation of 22 genetic resource management strategies a final tier should be considered, that of 23 landuse/geopolitical divisions.

    24 Keywords: Conservation genetics, Cedrela odorata, genetic differentiation,

    25 quantitative variation

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1 Introduction.

    2 The conservation of genetic resources within tree species is a recognised 3 necessity for their long term survival under changing conditions (Newton et al.,

    4 1999). However, current species delimitations often inaccurately describe 5 fundamental geographic and evolutionary units (Riddle and Hafner, 1999), whilst 6 population level approaches aimed at avoidance of inbreeding often fail to take 7 range-scale structure into account. For widespread species, the interaction of 8 gene flow (100’s of metres) and distribution (1000’s of metres), exposure to 9 diverse environmental regimes with differential selection pressures and 10 extinction / colonisation sequences creates structure on multiple scales. Therefore 11 to effectively conserve the genetic resources of a widespread species several 12 aspects of genetic variation need to be incorporated, i.e. identification of 13 conservation genetic units through integration of patterns of quantitative and 14 neutral genetic structure across multiple spatial scales. Once the organisation and 15 dynamics of genetic diversity are described, an approach that assesses species

    case-by-case, taking into account unique factors such as recommended forestry 16

    17 practice and geopolitical distribution, should allow formulation of an effective 18 strategy.

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    20 Current strategies for conservation of forest genetic resources typically involve 21 both in- and ex situ approaches, with in situ methods much more prevalent for

    22 non-cultivated species (Kanowski, 2000). Phylogeographic and population 23 genetic studies, using neutral molecular markers, provide a way of identifying in

    24 situ units and allow an understanding of diversity level and distribution, gene 25 flow routes and major genetic disjunctions within species. An approach that

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    1 combines assessments of evolutionary distinctiveness with population level 2 variation to quantify the contribution of evolutionary potential and population 3 level gene flow has been recommended as a basis for developing practical 4 measures (Petit et al., 1998). This approach has been demonstrated in a study of 5 Argania spinosa, an endangered Moroccan tree, that combined assessment of 6 phylogeographic structure and population genetics (El Mousadik, Petit, 1996a; El 7 Mousadik and Petit, 1996b). The analysis employed a combination of organellar 8 markers, to reveal phylogeographic structure, and isozymes, to estimate levels 9 and partitioning of allelic richness in populations. By collating evidence from 10 both sources, it was possible to identify evolutionarily distinctive conservation 11 units as well as prioritise populations within those units for protection. 12

    13 Whilst assessment of population genetic structure and phylogeographic patterns 14 provide a useful basis for deriving general conservation principles (Coates, 2000), 15 neutral genetic criteria cannot provide the whole picture (Paetkau, 1999). As a

    priority, describing the contemporary landscape of genetic variation should, 16

    17 where resources permit, consider quantitative variation. Population 18 differentiation for quantitative characters can indicate a genetic basis for 19 variation, and potentially an adaptive response to differing selection pressures. 20 Differentially adapted populations are clearly important resources that require 21 prioritisation as conservation units. Indeed assessment of quantitative variation 22 can highlight adaptively important discontinuities missed by neutral markers (e.g. 23 Bekessy et al., 2003).

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    1 This paper brings together, for the first time, results of previous studies on 2 chloroplast, total genomic and quantitative variation within the widespread 3 Neotropical tree Cedrela odorata L. We use these data to describe conservation

    4 units and assess the importance of these different types of information for 5 resource management and policy recommendation. Chloroplast DNA variation is 6 predominantly maternally inherited in Angiosperms (Harris and Ingram, 1991), 7 and appears to be in C. odorata (Cavers et al., 2003a). The chloroplast genome

    8 has a slow rate of mutation and lacks recombination, making intraspecific 9 variation phylogenetically interpretable. Hence cpDNA variation is useful for 10 examining historical colonisation processes and can shed light on the 11 evolutionary history of populations of a species. Random, dominant markers, like 12 AFLPs, can provide an estimation of the partitioning of genetic diversity at the 13 total genome level, although in practice, the source of such markers is 14 predominantly nuclear (Rieseberg, 1996). Whilst pollen and seed mediated gene 15 flow contribute to nuclear genetic structure, the former is considered more

    important due to the colonisation dynamics associated with seed dispersal. In 16

    17 addition, contemporary gene flow will erase historical population structure across 18 a species’ range, thus total genomic markers allow an assessment of the 19 contemporary genetic connectivity between populations of a species. Finally 20 divergence for quantitative traits, assessed through common garden experiments, 21 can identify genetic differences that may be associated with adaptive responses 22 to selection.

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    24 This study assesses the significance of differentiation for each of these three 25 types of genetic marker (organelle, whole genome and quantitative trait) using

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1 , Phi and Q respectively) for appropriate standard genetic parameters (i.e. GSTSTST

    2 populations across the range of C. odorata in Central America. Results are

    3 presented and weighted according to their perceived importance for prioritising 4 conservation units, and their value for conservation strategy is assessed. 5

    6 Case study: Cedrela odorata L.

    7 Spanish Cedar (C. odorata) is a Neotropical member of the hardwood family

    8 Meliaceae, well known for high quality timber. It is widespread, occurring

    9 naturally below 1200 m from around 25?N in Mexico, throughout the Caribbean 10 islands, lowland Central and South America to northern Argentina at 28?S. The 11 species is fast growing and light-demanding (Chaplin, 1980; Lamb, 1968; Valera, 12 1997) and may reach 40 m in height and 120 cm diameter. It is monoecious, 13 insect pollinated and has wind-dispersed seed. Due to significant over-14 exploitation, genetic erosion of the species has already occurred throughout its 15 natural distribution and trees of good form are now rarely found except in

    isolated areas (Styles and Khosla, 1976; Pennington et al., 1981). Continuing 16

    17 rapid deforestation in many parts of its range threatens remaining populations 18 (Bawa and Dayanandan, 1998).

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1 Methods.

    2 Results from three recent studies examining different aspects of genetic structure 3 in C. odorata were incorporated in the present study.

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    5 Seed sampled from 1980 trees, from 357 families in 30 populations from Central 6 America (Mexico to Panama, number of individuals and families sampled per 7 population varies between 24 and 132 and 2 and 22 respectively, Navarro, 2002) 8 were raised for quantitative study under common garden conditions in a 9 greenhouse, in a randomised block design. For each seedling the following 10 seventeen variables were measured: 62 days after sowing - (1) height, (2) length 11 and (3) width of the third leaflet from the tip of the leaf, on the third leaf from the 12 tip of the seedling, and (4) an index of leaflet shape (leaflet length divided by 13 leaflet width). The same four traits were measured 252 days after sowing (5-9) 14 and also (10) the length of the stem from the tip to the fourth branch in cm 15 (internodal distance), (11) the diameter in cm at 2 cm from the soil, (12) the

    number of leaflets per leaf, and (13) the class (tree quality, on a scale of 1 - 4), 16

    17 (14) weight of the leaves fresh and (15) weight of the leaves dry, (16) weight of 18 the branches fresh and (17) weight of the leaves dry. A standardised estimate of

    19 among-population differentiation was estimated using Q according to Wright ST

    20 (1951) and Merilä and Crnokrak (2001).

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    22 Variation in the chloroplast genome was assessed in 580 individuals sampled 23 from 29 populations throughout Mesoamerica (10 in Costa Rica, 3 in Panama 24 and 4 in each of Mexico, Guatemala, Honduras and Nicaragua, Cavers et al.

    25 2003a). Phylogeographic analysis consisted of screening variation at two cpDNA

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1 loci, using universal primers and protocol modifications as detailed in Cavers et

    2 al. (2003a). Total (h) and within-population diversity (h) and level of TS

    3 population subdivision (G) was calculated using the program HAPLONST, ST

    4 available at http://www.pierroton.inra.fr/genetics/labo/Software (Pons and Petit,

    5 1996).

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    7 Nine Costa Rican populations (with a total of 121 individuals sampled) were

    8 screened for variation at 145 AFLP fragments, as detailed in Cavers et al.

    9 (2003b). The data were analysed for structure using Analysis of Molecular

    10 Variance (AMOVA, WINAMOVA 1.5 Excoffier et al., 1992; Miller, 1997), with 11 a pairwise genetic distance matrix based on shared presence of fragments (Huff

    12 et al., 1993). A Neighbour-Joining tree was prepared, based on pairwise ; ST

    13 estimates (derived from AMOVA) between all populations. 14

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1 Results.

    2 A major disjunction (Figure 1, average Q for all populations was 0.34 ? 0.02) ST

    3 was identified for quantitative genetic characters that distinguished populations 4 collected in Yucatan/Honduras/Nicaragua from those collected in Panama. Costa 5 Rican populations were split, with populations from the northwest of the country 6 clustered with the Yucatan/Honduras/Nicaragua group and all others (from the 7 southwest and east) clustered with the Panama group (Navarro 2002).

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    9 In the phylogeographic analysis of cpDNA variation, five haplotypes were 10 characterised in three geographically differentiated lineages (Figure 2): 11 designated Northern (Mexico, Guatemala, 2 haplotypes), Central (Honduras, 12 Nicaragua, northwestern Costa Rica, 1 haplotype) and Southern (east and 13 southwest Costa Rica, and Panama, 2 haplotypes). The three lineages are 14 differentiated from each other by three mutations or more, with the Northern and 15 Central lineages most genetically distant from each other (9 mutations, Figure 2).

    Both Northern and Southern lineages contain two haplotypes separated by single 16

    17 mutations. Almost all of the populations were fixed for a single haplotype (only 3 18 of the 29 populations showed within-population diversity) and the global level of 19 population subdivision as measured by G was 0.96 (Cavers et al. 2003a). ST

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    21 Within Costa Rica, AFLP analysis identified differentiation between populations 22 from the northwest region and those from the east / southwest of the country as 23 the principal source of variation (83.47%, Figure 3). However, further 24 subdivision was present within the latter subgroup, with 52.61% of variation 25 within this group partitioned between populations. This between-population

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    1 structure was due primarily to differentiation between populations from the east 2 and the southwest of the country: in other words, on either side of Costa Rica’s

    3 central mountain ranges (Cavers et al. 2003b).

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    5 Overall quantitative variation clearly delineates the species into a northern and 6 southern grouping with a sharp distinction between the two, but with no other 7 grouping evident (Figure 1, Navarro 2002). Chloroplast DNA analysis reveals a 8 strong phylogeographic structure at the rangewide scale, with three lineages 9 distributed as geographically exclusive units (Cavers et al. 2003a). The

    10 divergence between the Southern lineage and those in the north (Northern and 11 Central, Figure 2) coincides exactly with the divergence seen for quantitative 12 traits. However, the deepest divergence in the cpDNA data (between Northern 13 and Central lineages) was not observed using quantitative measures. AFLP 14 analysis, although restricted to Costa Rican populations, also found significant 15 differentiation between populations possessing the Central and Southern cpDNA

    lineages (Cavers et al. 2003b). An initial investigation of AFLP variation in 16

    17 Yucatan populations of C. odorata (Cavers unpublished) suggests that material

    18 from this region clusters most closely with that from northwestern Costa Rica in 19 line with observations from quantitative data (Figure 3). However, AFLP 20 analysis identified further significant population structure, between eastern and 21 southwestern Costa Rican populations, i.e. within the Southern cpDNA lineage. 22

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