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Population Biology |
2Laboratorio de Biodiversidad Molecular y Banco de ADN, Jardín Botánico Canario Viera y Clavijo, Ap. de correos 14 de Tafira Alta, 35017 Las Palmas de Gran Canaria, Spain; 3Jardín de Aclimatación de La Orotava (ICIA), C. Retama 2, 38400 Puerto de la Cruz, Tenerife, Spain; 4Casa Sick-Esquinzo, 35626 Jandía, Fuerteventura, Spain
Received for publication September 2, 2005. Accepted for publication May 6, 2006.
ABSTRACT
We examined data for 11 allozyme loci in 14 populations that represent the distribution of the endangered Lotus kunkelii, the narrowly distributed L. arinagensis (both endemic to Gran Canaria), and the broad-ranging L. lancerottensis (endemic to the easternmost Canary Islands, Fuerteventura and Lanzarote) to explore and construe patterns of genetic variation and use this data to assess the controversial taxonomic status of L. kunkelii relative to L. lancerottensis. While L. kunkelii maintains low levels of variation, presumably as a consequence of prolonged inbreeding due to very low population size and sharp geographic isolation, the other two taxa have much higher indicators of polymorphism than those reported for other oceanic island endemics. Lotus arinagensis has the highest genetic polymorphism and the lowest interpopulation differentiation, presumably because of its considerable antiquity and habitat stability, despite recent fragmentation. The high interpopulation differentiation in L. lancerottensis is attributed to the Atlantic acting as a barrier, reducing gene flow within islands. Evolutionary analysis of the allozyme evidence indicates that L. kunkelii is genetically closer to L. arinagensis than to L. lancerottensis, thereby dispelling the taxonomic uncertainty and supporting L. kunkelii as a distinct species, warranting legal protection in the forthcoming catalog of threatened Canarian species.
Key Words: allozymes • Canary Islands • conservation • endemics • evolutionarily significant units • genetic divergence • Lotus • taxonomy
Morphological characters offer variation that has proven useful to assess evolutionary relationships or to establish unambiguous taxonomic circumscriptions in most plant lineages. However, this variation is difficult to handle statistically because it is mostly attributable to the combined action of many different loci (Doebley et al., 1990
; Koornneef et al., 2004
), whose inheritance and exact contribution to the phenotype are unknown. In addition, environmentally induced variation may be confused easily with genetically based (i.e., systematically meaningful) variation, and this difficulty may sometimes undermine the value of evolutionary or taxonomic inferences based only on morphological traits. These intrinsic limitations notwithstanding, when morphology cannot provide unambiguous diagnostic characters, the taxonomist is hindered by lack of useful characters, thereby begetting controversies that make the delimitation of operative taxonomic units (OTUs) difficult. In these cases, it is of utmost importance to add variables other than the morphological to facilitate an objective classification, especially when the organisms are endangered and their taxonomic rank may affect their legal protection status.
Lotus kunkelii (Esteve) Bramwell & Davis and L. lancerottensis Webb & Berthel. are two taxa of Lotus sect. Pedrosia endemic to the eastern Canary Islands. While L. kunkelii is known from only one extremely small population (less than 50 individuals according to Bañares et al., 2003
) on the eastern coast of the island of Gran Canaria, L. lancerottensis is widespread on Fuerteventura and Lanzarote, where it shows considerable ecological variation (Fig. 1). The taxonomic position of L. kunkelii and L. lancerottensis has stirred controversy ever since the former taxon was described by Esteve-Chueca (1972)
. This author treated L. kunkelii as a subspecies of L. lancerottensis (L. lancerottensis subsp. kunkelii Esteve) based on several morphological traits. However, Bramwell and Davis (1972)
contend that L. kunkelii should be treated as a distinct species. Because no other data have been examined for these two taxa until now, the taxonomic status dispute has remained unsettled.
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) argues that the threshold between subspecific and interpopulation differentiation is ambiguous, and establishes that the minimum unit for legal protection should be the species. Although this is an arbitrary decision not supported by science, solving the taxonomic dispute that affects L. kunkelii is of clear conservation interest because its consideration as a species or as a subspecies of L. lancerottensis will determine, respectively, its inclusion in the lists of protected Canarian endemics or its exclusion from this instrument of legal protection.
The area where this population lives was first protected by the Canarian Environmental Law under the designation of "natural landscape of interest" (Boletín Oficial de Canarias, 1987
), which was later renamed as a "site of scientific interest" (Boletín Oficial de Canarias, 1994
). Despite this legal protection, recent anthropogenic pressures in the area of distribution of L. kunkelii have brought about enhanced habitat degradation and a swift decline in the number of individuals, which greatly increase the threat of endangerment.
The discrete data on genetic variation afforded by allozymes are more amenable to mathematical analysis than are morphological traits and have often been used with success as surrogates for quantitative genetic variation. Although in recent meta-analyses (Reed and Frankham, 2001
) a low correlation between molecular and quantitative variables has been found, allozymes have so far provided valuable ancillary information to clarify taxonomic issues in a number of plant lineages (Elisiário et al., 1999
; Cabrita et al., 2001
), including other Canarian Lotus within sect. Pedrosia (Oliva-Tejera et al., 2005
). Therefore, we consider this molecular technique a proper first choice to address the taxonomic issue that affects L. kunkelii and L. lancerottensis so that we can understand their pattern of genetic divergence. This approach will set the stage for improving our knowledge of the evolution and taxonomy of the group.
Our specific objectives in this investigation were (1) to explore and construe the patterns of evolutionary divergence in L. kunkelii and L. lancerottensis using allozyme variation and (2) to use this molecular information as a tool for assessing the protection status of L. kunkelii. To gauge the extent of genetic differences between L. kunkelii and L. lancerottensis, we include L. arinagensis Bramwell (a well-delimited species within sect. Pedrosia that is distributed exclusively along the eastern coast of Gran Canaria) as an external reference.
MATERIALS AND METHODS
Plant material
Lotus lancerottensis (2n = 14; Larsen, 1956
) is a perennial plant covered with silky appressed hairs. Its stems are prostrate or ascendant, subglabrous, with sessile five-foliolate leaves. The leaflets are obcordate to obovate, very rarely succulent. Inflorescences consist of peduncles of 2–4 cm each, the teeth are always longer than the tube, and legumes are smooth. By contrast, L. kunkelii (2n = 28, Ortega, 1976
) possesses a densely hirsute stem with patent hairs, and its leaves are densely hairy and much smaller than those of L. lancerottensis. The leaflets are always succulent, obcordate to rounded, the calyx teeth are the same length as the tube or shorter, and the legumes are rough. Lotus arinagensis [2n = 28; Ortega, 1976
, as L. leptophyllus (Lowe) K. Larsen] is a slow-growing perennial, with densely tomentose (white) prostrate stems. Its leaves are sometimes three-foliolate, and leaflets are ovoid, silvery, and subsucculent. The inflorescences have peduncles of 1–1.5 cm, and legumes are smooth.
Sampling
In all cases, the sampling design followed Caujapé-Castells (2004)
and included all the known areas of population distribution in order to estimate consistently the levels of genetic variation. The only known population of the endangered L. kunkelii occurs at the Barranco de Jinámar (Gran Canaria), and its estimated size is less than 50 individuals according to the latest census (Bañares et al., 2003
). In this specific case, sampling was restricted to the individuals whose size was enough to ensure plant survival after leaf detachment. In the broad-ranging L. lancerottensis, we sampled 220 individuals from 10 populations (five from Lanzarote and five from Fuerteventura). In the narrowly distributed L. arinagensis, we sampled 143 individuals from the three known populations in Gran Canaria. Sample sizes (Fig. 1) are strictly related to the size of target populations and ranged from eight individuals in population LKJIN (L. kunkelii) to 98 in LAARI (L. arinagensis). In all cases, leaf samples from individual plants were deposited in numbered, zippered plastic bags that were then refrigerated in a portable cooler until storage in ultralow freezers at the Jardín Botánico Canario "Viera y Clavijo" (JBCVC) in Gran Canaria, where they remained until used for extract preparation. The time from leaf collection to storage in the ultralow freezers was under 4 d in all cases; longer times would have entailed a loss of activity for most assayed enzymes (F. Oliva-Tejera, Jardín Botánico Canario "Viera y Clavijo", unpublished data).
Electrophoretic analysis and data processing
Allozyme electrophoreses and interpretations followed, respectively, Caujapé-Castells et al. (2001)
and Oliva-Tejera et al. (2005)
and allowed us to score six enzymes: phosphoglucomutase (PGM, E.C. 5.4.2.2), isocitrate dehydrogenase (IDH, E.C.1.1.1.42), esterase (EST, 3.1.1.1), phosphogluconate dehydrogenase (6PGD, E.C.1.1.1.44), malate dehydrogenase (MDH, E.C.1.1.1.37), and malic enzyme (ME, E.C.1.1.1.40). All gel interpretations were drawn in the matrix provided by the computer program Transformer-3 (Caujapé-Castells and Baccarani-Rosas, 2005
), which was also used to generate all the input files needed to analyze these data using different population genetic softwares.
Number of alleles per locus (A), percentage of polymorphic loci (P) [a locus was considered polymorphic if more than one allele was detected], Ho (i.e., average heterozygosity based on direct counts), He (i.e., average proportion of heterozygotes based upon Hardy–Weinberg expectations), inbreeding coefficients [FIS = 1 – (Ho/He) ] (Hartl and Clark, 1989
), and genetic identities (INEI,; Nei, 1978
) were calculated using BIOSYS-1, version 1.7 (Swofford and Selander, 1989
) at the species and population levels from genotype data corresponding to each locus. INEI was calculated for comparative purposes with other studies with congeneric Canarian endemics that use this index to quantify the extent of genetic divergence. Popgene, version 1.32 ( Yeh et al. 1997
), was used to calculate the average effective number of alleles per locus (Ae) following the formula Ae = 1 + 4Neµ (Kimura and Crow, 1964
) and to carry out Ewens–Watterson homozygosity tests of neutrality (Watterson, 1978
).
The values of Rogers' genetic distance (Rogers, 1972
) between population pairs were used to build a cluster with the neighbor joining (NJ) algorithm (Fig. 2). Rogers' distance is more sensitive to disjunct or private alleles than other measures of genetic distance that use an overall allele frequency; therefore, it is generally believed to be more appropriate to address cases of presumably recent evolutionary divergence (Rogers, 1991
; Britten and Brussard, 1992
). The NJ algorithm allows for unequal rates of molecular evolution and is thus more likely to reflect evolutionary relationships than other clustering methods.
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) and BIOSYS-1, version 1.7 (Swofford and Selander, 1989
). To evaluate the genetic differences among L. kunkelii and the other two taxa, F statistics were also calculated for the artificial assemblages (L. kunkelii + L. arinagensis, L. kunkelii + L. lancerottensis and L. arinagensis + L. lancerottensis). The rationale of this procedure is to use the eventual changes in the values of FST to assess whether the addition of the population of L. kunkelii disrupts significantly the genetic cohesion of L. lancerottensis (in which case L. kunkelii should be considered a species on its own) or not (and then it would be feasible to consider L. kunkelii as a disjunct population of L. lancerottensis). The values of FST for L. arinagensis alone and in combination with the other two taxa were used to calibrate the results obtained for L. kunkelii and L. lancerottensis.
We applied a sign test for heterozygosity excess (Cornuet and Luikart, 1996
) to detect whether populations had experienced recent historical bottlenecks. This test compares expected heterozygosity (He) under Hardy–Weinberg equilibrium to the heterozygosity expected at mutation–drift equilibrium (Heq) in a sample that has the same size and the same number of alleles as the sample used to measure He (Luikart and Cornuet, 1998
). Because low frequency alleles are lost at a much faster rate than heterozygosity in a bottleneck situation, bottlenecked populations are expected to have a heterozygote excess. Calculations were made based on allele frequency data under the Stepwise Mutation Model (SMM) and the Independent Allele Model (IAM) using the program Bottleneck-PC (Piry et al., 1998
).
RESULTS
Genetic interpretations of the six enzymes resolved allowed us to score 11 putative loci, none of which was monomorphic throughout the populations. Of the 47 alleles scored (Table 1), one was exclusive to the endangered L. kunkelii (Idh1-e), four to the broad-ranging L. lancerottensis (Est1-a and Pgm3-a to Lanzarote, and Pgm2-b and 6Pgd2-b to Fuerteventura), and five to the narrowly distributed L. arinagensis (Idh1-a, Idh1-b, Pgm2-a, Pgm3-b, 6Pgd4-e) (Table 1). We detected two alleles shared exclusively between L. kunkelii and L. arinagensis (Est1-b, Est1-e), one between L. arinagensis and L. lancerottensis (Est1-h) and none between L. kunkelii and L. lancerottensis. The remaining 34 alleles were shared by different combinations of the 14 populations analyzed. We did not detect diagnostic alleles (i.e., alleles that were monomorphic in one taxon and not present in the others).
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genetic distance resulted in a single tree (Fig. 2), that placed L. kunkelii closer to L. arinagensis than to L. lancerottensis. However, two populations of L. lancerottensis from Fuerteventura (LLVMB and LLJAN) clustered with the populations of L. arinagensis and L. kunkelii.
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for other Canarian endemic taxa of Lotus, but any artificial assemblage with L. kunkelii resulted in INEI far lower than expected for conspecific taxa according to that paper. The values of FST also increased substantially when L. kunkelii was added to form artificial assemblages with the other two taxa; while the highest FST was detected in L. lancerottensis + L. kunkelii (FST = 0.215, vs. FST = 0.189 for L. lancerottensis alone), the highest increase in the value of this parameter corresponded to L. arinagensis + L. kunkelii (FST = 0.162, vs. FST = 0.083 for L. arinagensis alone). DISCUSSION
Levels and apportionment of genetic variation
These taxa of Lotus maintain substantial levels of genetic heterogeneity, consistent with the emerging picture of higher variation in Canarian endemics relative to those from other oceanic archipelagos (Francisco-Ortega et al., 2000
). Their basic indicators of polymorphism (Table 2) are in all cases higher than the average values calculated by Hamrick and Godt (1989)
for endemic plants (A = 1.39, P = 26.3, He = 0.063). However, Helenurm (2001)
detected much higher levels of polymorphism in Jepsonia malvaefolia Small (Saxifragaceae), an endemic to the Channel Islands of California and Guadalupe Island (Mexico), with averages of A = 2.9, P = 95.2, He = 0.179. Overall, the values for the Lotus species analyzed in this paper are slightly higher than those reported for the Gran Canarian pine forest endemics L. holosericeus Webb & Berthel. and L. spartioides Webb & Berthel. (Oliva-Tejera et al., 2005
). The endangered L. kunkelii also contains more variation than some populations analyzed by Oliva-Tejera et al. (2005)
. The values of these parameters for the broad-ranging L. lancerottensis and the narrowly distributed L. arinagensis are only slightly lower than those detected by Gauthier et al. (1998)
in the mainland diploid species L. alpinus (Schleich. ex DC) Ramond (averages A = 2.8, Ho = 0.219, He = 0.279). The degree of genetic variation within these taxa, as measured by the average population diversity (Hs, Table 3), is in all cases much higher than the averages published for plants from Hawaii (Hs = 0.064, DeJoode and Wendel, 1992
) or the Juan Fernández islands (Hs = 0.042, Crawford et al., 2001
), and almost two-fold the average value published for Canarian taxa (Hs = 0.137, Francisco-Ortega et al., 2000
).
The skew in sample sizes associated with the increasing rarity of populations and taxa is one additional factor that may have affected the probability of finding genetic heterogeneity, thereby decreasing the values of the indicators of genetic variation in small populations. While it is important to consider this aspect for the thorough discussion of results, it does not seem to preclude our findings. The populations LALMF and LASAL (L. arinagensis), that are about the same size as that of the endangered L. kunkelii, have indicators of genetic polymorphism much higher than L. kunkelii (Table 2). Therefore, the levels of genetic variation detected in these extremely small populations of L. arinagensis and L. kunkelii seem to be related more to the contrasting historical and biological features of the corresponding taxa than to their low sample size.
Lack of evidence for genetic bottlenecks in the three taxa analyzed (Table 2) and the positive FIS values (Table 3) suggest that these high levels of genetic variation have been attained in a context of environmental stability and despite an overall predominance of inbreeding. As hypothesized by Oliva-Tejera et al. (2005)
, seed dispersal through barochory (i.e., gravity dispersal) in Lotus must be a paramount factor to explain the observed heterozygote deficit in most populations because it fosters small genetic neighborhoods where reproduction takes place between related individuals. Furthermore, because our sampling design attempted to represent the distribution of individuals within the population area, the distances between sampled plants in sizeable populations were often large and may possibly have resulted in a Wahlund effect (Wahlund, 1928
), thus contributing to the high FIS values. However, only prolonged inbreeding (and not a Wahlund effect) is the most feasible explanation for the high FIS values in L. kunkelii and in LASAL and LALMF (the two smallest populations of L. arinagensis) because our sampling in these populations represents a high proportion of the visible individuals.
Basic population genetics theory predicts a dramatic loss of genetic variation in small populations due to an enhanced action of drift and inbreeding (Barrett and Kohn, 1991
). Therefore, the genetic depauperation of L. kunkelii could be construed as a consequence of its demographic traits and of its sharp geographic isolation (it occurs on a secluded slope of a cliff facing the sea at the east of Gran Canaria). Although the bottleneck test was not significant for this population (data not shown), we consider it likely that reductions in population size due to recent anthropogenic disturbances might have affected its levels of variation. Another likely consequence of isolation and low population size in L. kunkelii is an increased frequency of inbred matings, as suggested by its value of FIS, the highest of the three taxa examined (Table 2).
In L. arinagensis , the sharp differences in population size among LASAL, LALMF, and LAARI (Fig. 1), the fact that the genetic variation of the smallest populations (LALMF and LASAL) is not a strict subset of that in LAARI (Table 1), gravity seed dispersal (that tends to foster reproduction in family clumps) and the homogeneous environmental conditions in its area of distribution hint that the interpopulation genetic homogeneity observed in this taxon is likely a result of recent fragmentation.
By contrast, interpopulation differentiation in the broad-ranging L. lancerottensis (Table 3) is more than two-fold that in L. arinagensis. At the morphological level, this within-island heterogeneity is illustrated by the population LLJAN from Fuerteventura, that was originally described as Lotus erythrorhizus Bolle (Bolle, 1891
), later considered by Brand (1898)
as Lotus glaucus Ait. var. erythrorhizus (Bolle) Brand, and finally established as a variety of Lotus lancerottensis by Kunkel (1976)
. At odds with these independent morphological assessments, no substantial difference between LLJAN and the other populations in terms of rare alleles was detected. Although this population has the lowest genetic identity with other populations within Fuerteventura (INEI = 0.908 with LLCOR, Table 4), this value is within the range expected for conspecific populations. In the absence of evidence for local adaptation, the most likely hypothesis to explain within-island differentiation in L. lancerottensis is that genetic differentiation among these populations was a by-product of time since genetic isolation and attendant stochastic forces.
Between islands, the two exclusive alleles detected in Fuerteventura and in Lanzarote (Table 1) probably reflect the contribution of mutation to the observed geographic pattern. Drift is another potentially important factor to explain genetic heterogeneity in oceanic islands (Crawford et al., 1987
) and, in the hypothesized context of a long divergence time and low levels of gene flow, it probably played a substantial role in the enhancement of inter-island genetic differentiation in L. lancerottensis.
The genetic divergence between L. lancerottensis and L. kunkelii
In order to discuss the extent of evolutionary divergence of the endangered L. kunkelii and the broad-ranging L. lancerottensis, we will first assess if these two taxa fulfill the definition of an "evolutionarily significant unit" (ESU, Waples 1991
): a population or group of populations that (1) is reproductively isolated from other con-specific populations, and (2) represents an important component of the evolutionary legacy of the species. Because ESUs are widely equated with conservation units (Moritz, 1994
; Karl and Bowen, 1999
), this conceptual framework seems especially well suited to substantiate eventual decisions to protect L. kunkelii. Only if L. kunkelii and L. lancerottensis can be considered independent ESUs are we then justified to evaluate if their degree of genetic divergence is enough to recognize L. kunkelii at species rank or the same taxon as a subspecies of L. lancerottensis.
To test the degree of reproductive isolation between these taxa, it seems adequate to use the values of FST (Wright, 1951
), because this parameter accounts for the proportion of total variation that can be explained by the differentiation among populations and, therefore, reflects the role of gene flow as a force of genetic cohesion. The second part of the definition of ESU is more difficult to assess quantitatively given the diversity of biological or historical traits that can be interpreted as "evolutionary legacy." According to Waples (1995)
, this term refers to "the genetic variability that is a product of past evolutionary events and that represents the reservoir upon which future evolutionary potential depends." Because rare alleles may confer survival capabilities to populations after an environmental contingency (Schonewald-Cox et al., 1983
), they are important components of the future evolutionary potential of populations, and we used them as indicators of evolutionary legacy in these three taxa of Lotus.
Population genetics theory predicts that the population groups where FST values are higher can be considered to be genetically more heterogeneous, presumably as a consequence of the discontinuation of genetic interchange among the constituent populations (Slatkin, 1985
, 1987
, 1994
). By contrast, higher levels of gene flow among populations should be assumed in groups of populations that have lower FST values (Slatkin, 1985
). The quantitative insight offered by this parameter shows that the inclusion of L. kunkelii in any artificial assemblage with the other two taxa examined induces a substantial increase in the values of FST (FST = 0.215 for L. kunkelii + L. lancerottensis, FST = 0.162 for L. kunkelii + L. arinagensis, and FST = 0.205 for L. lancerottensis + L. arinagensis). This evidence indicates that the taxonomic inclusion of L. kunkelii within either L. lancerottensis or L. arinagensis would disrupt the genetic cohesion of these two taxa severely and that population genetic differentiation between L. lancerottensis and L. kunkelii is even higher than that between L. lancerottensis and L. arinagensis, whose taxonomic position as different species is undisputed. Qualitatively, although we did not detect substantial "evolutionary legacy" in L. kunkelii (only one exclusive allele, Table 1) this taxon shares more alleles with L. arinagensis (20 of 21) than with L. lancerottensis (15 of 21) (Table 1). These two genetic results bolster the consideration of L. kunkelii and L. lancerottensis as independent ESUs according to Waples' (1991) definition.
Most relevant for assessing the extent of genetic divergence between L. kunkelii and L. lancerottensis, the average genetic identity between these two taxa (INEI = 0.823) is far lower than that detected level for any other pair of Canarian Lotus species examined with allozyme electrophoresis (INEI = 0.938 between L. spartioides and L. holosericeus, Oliva-Tejera et al., 2005
), and even lower than the average INEI between L. kunkelii and L. arinagensis (INEI = 0.886). Furthermore, the minimum values of INEI were detected between L. kunkelii and some populations of L. lancerottensis (Table 4). Although the allozyme genetic evidence compellingly suggests that L. kunkelii is much closer to L. arinagensis than to L. lancerottensis (Fig. 2), it would be incorrect to infer from our results that L. kunkelii and L. arinagensis should be merged into a single ESU because they are distinct both genetically and morphologically; the rationale of including L. arinagensis in these analyses was to provide an external reference to gauge the genetic differences between L. kunkelii and L. lancerottensis, which were the only source of taxonomic discrepancies. Overall, these comparisons indicate that the range of genetic divergence between L. kunkelii and L. lancerottensis corresponds to that expected for different species of Canarian Lotus.
Conclusion
High genetic variation levels and low interpopulation differentiation in L. arinagensis are probably a consequence of a considerable antiquity, habitat stability, and only recent fragmentation. By contrast, L. lancerottensis has a substantial interpopulation differentiation, which is best construed as the effect of prolonged action of time since isolation and stochastic forces to increase genetic differentiation both within and between its islands of distribution. Lotus kunkelii possesses the lowest levels of variation detected, probably due to increased inbreeding brought about by geographic isolation and by reductions in population size associated with anthropogenic disturbance and recurrent bottlenecks. Overall, our results consistently show that (1) the extent of evolutionary divergence between the endangered L. kunkelii and the broad-ranging L. lancerottensis is much higher than that detected between either L. lancerottensis and the narrowly distributed L. arinagensis or between other Canarian Lotus examined with allozymes (Oliva-Tejera et al. 2005
) and that (2) at odds with Esteve-Chueca's (1972) taxonomic proposal, L. kunkelii is genetically closer to L. arinagensis than to L. lancerottensis. These conclusions agree with the Bramwell and Davis (1972)
classification and support the consideration of L. kunkelii as a distinct species, whose extremely endangered population should be given maximum protection according to the new methodology for the catalog of threatened Canarian species (Martín-Esquivel 2004
).
FOOTNOTES
1 The authors thank D. Bramwell, B. Navarro, and J. Naranjo (at the Jardín Botánico Canario "Viera y Clavijo"); J. R. Acebes-Ginovés (at the Departamento de Botánica of the Facultad de Farmacia in the Universidad de La Laguna, Tenerife) for comments and insight on an earlier version of this manuscript; staff of BIOTA-genes at the Centro de Planificación Ambiental of the Gobierno de Canarias in Tenerife for their trust and stimulus; and the Cabildo de Gran Canaria for institutional support. This investigation was funded by the European initiative INTERREG IIIB-Atlántico and the Gobierno de Canarias to the project BIOTA-genes. This paper was written while J. C. C. was a "Ramón y Cajal" researcher in molecular population genetics and phylogenetics at the JBCVC. J. C. C. thanks the Ministerio de Educación y Ciencia and the Cabildo de Gran Canaria for their support. 
5 Author for correspondence (julicaujape{at}grancanaria.com
)
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