Conserv Genet DOI 10.1007/s10592-011-0277-y RESEARCH ARTICLE Distinctiveness, rarity and conservation in a subtropical highland conifer Alicia Mastretta-Yanes • Ana Wegier • Alejandra Vázquez-Lobo • Daniel Piñero Received: 29 June 2011 / Accepted: 20 September 2011  Springer Science+Business Media B.V. 2011 Abstract Juniperus blancoi is a rare subtropical conifer with a wide yet restricted distribution and three recognized varieties. In this study, its ecological and genetic exchangeability are tested based on morphological descriptions, habitat differences, size measures, growth form, population genetics estimates and phylogeography, using the trnC-trnD plastid DNA region. Populations show differences in their habitat and morphological characteristics. Phylogeographic structure indicates a complex evolutionary history of expansion, fragmentation, and isolation processes. This resulted in high haplotype diversity (h = 0.863) and differentiation values (Dest = 0.866 and Dest [ 0.5 in most pairwise comparisons) and a clear geographic structure with well defined groups. As a consequence, although the species has only 8 known populations, it must be divided into at least five distinct conservation units. Thus protecting a rare species Electronic supplementary material The online version of this article (doi:10.1007/s10592-011-0277-y) contains supplementary material, which is available to authorized users. A. Mastretta-Yanes (&)
A. Wegier
A. Vázquez-Lobo
D. Piñero Departamento de Ecologı́a Evolutiva, Instituto de Ecologı́a, Universidad Nacional Autónoma de México, Apartado postal 70-275, Mexico, DF 04510, Mexico e-mail: ticatla@gmail.com Present Address: A. Mastretta-Yanes Centre for Ecology, Evolution and Conservation, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK Present Address: A. Wegier CENID-COMEF, Instituto de Investigaciones Forestales Agrı́colas y Pecuarias, Mexico, DF 04010, Mexico could imply the conservation of a complex evolutionary history of non-exchangeable populations. Keywords Rarity
Evolutionary significant units
Conservation units
Exchangeability
Juniperus blancoi
trnC-trnD
cpDNA Introduction It is common to use the words conservation and rarity together. Usually, rare species are subjected to conservation strategies because they represent some curiosity or because they are under a greater threat than widely distributed taxa (Bevill and Louda 1999; Soltis and Gitzendanner 1999), but they can also be a natural product of the evolutionary processes affecting species. Rabinowitz (1981) delimited seven different forms of rarity distinguished by dichotomous differences in local abundance (high-low), geographic range (narrow-wide), and habitat specificity (wide-narrow). Such a scheme accurately describes rarity in a spatial context, but we must consider the temporal framework of rarity (Harper 1981): what is rare now may not have been before and vice versa. Understanding rarity as an evolutionary issue implies that species became rare by several means and with different ecological and evolutionary implications. One of these routes suggests that some species are rare because they could have passed through a more pronounced process of fragmentation and isolation than other species (Rabinowitz 1981; Fiedler and Ahouse 1992; Kunin and Gaston 1993; Bevill and Louda 1999). This coincides with the finding that many ecosystems rich in rare species are often found in isolated climates that potentially were more common during glacial periods but became fragmented during interglacials (Ohlemüller et al. 2008). 123 Conserv Genet Within Mexico’s temperate forests 25% of Juniperus world taxa are present, and 58% of these are endemic (Adams 2008). Many of these junipers and other Mexican conifers are rare species reported from a few isolated populations or from only one locality (Farjon and Styles 1997; Price et al. 1998; Adams 2008). Juniperus blancoi Martı́nez is a rare species endemic to Mexico. It is in the Vulnerable D2 category according to the IUCN Red List of Threatened Species (version 2.3; IUCN 2010), which means that it is facing a high risk of extinction in the wild because its populations are restricted in area and in number. However, so far it has not been included in the Mexican NOM-059SEMARNAT-2010 list of endangered species. Three varieties are recognized within the species: J. blancoi Martı́nez var. blancoi; J. blancoi Martinez var. mucronata (R.P. Adams) Farjon, and J. blancoi Martinez var. huehuentensis R. P. Adams, S. González and M. González Elizondo (Fig. 1). The species occurs in the Sierra Madre Occidental (SMO) and Trans Mexican Volcanic Belt (TMVB) mountain ranges. This means that its distribution range is from Northwest to Central Mexico (Fig. 2a), which corresponds to more than 1,150 km separating its northern and southern distribution limits (Table 2), but only eight localities (Table 1) occupying a total area of less than 10 km2. As with other rare conifers (Molina-Freaner et al. 2001; Cuenca et al. 2003) this distribution suggests that the species would need special conservation strategies that consider variation at the population level. Conservation below the species level has been broadly discussed, determining the importance of creating conservation units (CUs) within a species (Ryder 1986; Waples 1991; Dizon et al. 1992; Avise 1994; Vogler and DeSalle 1994; Crandall et al. 2000; Fraser and Bernatchez 2001). Also, the pragmatic urge to protect irreplaceable units of biodiversity, called Designatable Units, from becoming extinct or extirpated from a jurisdiction has been stated by Green (2005). Conservation units are mostly defined using Evolutionary Significant Units (ESUs), a population unit that merits separate management and has a high priority for conservation (Ryder 1986). Fraser and Bernatchez (2001) reviewed different methodologies to define them and concluded that designating ESUs should be done flexibly from a practical standpoint on a case-by-case basis, remembering the overarching conservation goals when confronted with a lack of sufficient data. They defined ESUs as a lineage demonstrating highly restricted gene flow from other lineages within the higher organizational level (lineage) of the species, stating that to maximize the probability of protecting the genetic variance within a species it is necessary to consider both the historical and the ecological evolutionary forces that gave rise to the isolated lineages. Crandall et al. (2000) presented an operational criterion for determining the genetic and ecological distinctiveness of populations in terms of rejection of the null hypotheses, both recent and historical, of genetic and ecological exchangeability. Genetic exchangeability is the factor that defines the limits of spread of new genetic variants through gene flow. It is rejected when there is evidence of restricted gene flow between populations obtained from molecular markers or the existence of geographic barriers (Crandall et al. 2000). The central idea of ecological exchangeability is that individuals moved between populations will occupy the same ecological niche or selective regime. The null hypothesis of ecological exchangeability is rejected when there is evidence of population differentiation in heritable traits of life history, morphology, habitat, and quantitative trait loci, owing either to natural selection or genetic drift (Crandall et al. 2000). In this study, we describe J. blancoi’s rarity and its populations’ characteristics in order to test whether or not they are ecological and genetically exchangeable, following Crandall et al.’s (2000) proposal. CUs were defined based on a pragmatic standpoint to delimit irreplaceable units within the species (Fraser and Bernatchez 2001) and on the urgency to recognize if they need protection (Green 2005). This is important for the sake of conservation of the species, and relevant to the study of the evolutionary history of the endemic-rich forests it inhabits. Fig. 1 Species habitat and morphological variation. a J. blancoi var. blancoi, trees [20 m high growing close to a stream in a humid glen in La Preciosita. b J. blancoi var. huehuentensis, a prostrate shrub form at the summit of Cerro Huehuento. c J. blancoi var. mucronata, shrubs and trees 3–9 m high growing in rocky riverside 123 Conserv Genet Fig. 2 Geographic distribution of genetic variation of J. blancoi (trnC-trnD cpDNA) across the species range. a Haplotype distribution and frequency in J. blancoi populations: i Yécora; ii Cerro Mohinora; iii Cerro Huehuento; iv El Salto; v Rı́o Patitos; vi Presa Brockman; vii San José del Rincón and viii La Preciosita. See Table 1 for J. blancoi varieties. Haplotypes color coding as in (b). Hydric regions are shown as filled areas, biogeographical regions are delimited with lines: SMO Sierra Madre Occidental and TMVB Trans Mexican Volcanic Belt. b Haplotype network showing hypothesized relationships among haplotypes. The circle size indicates the relative frequency of each haplotype. Missing haplotypes are represented as open circles. The kind of mutation of each mutational step is indicated as: * substitution;  inversion and u indel, details in Online Resource 1. c Phylogenetic relationships between J. blancoi haplotypes (H1– H10), J. scopulorum (J.s. 10933, 10935) and J. virginiana (J.v. 10220, 10221) constructed using MP Bootstrap values based on 1,000 repetitions are above the branches Methods height and diameter. General environmental conditions and land use were established with field observations and CONABIO (1999) data. Tissue samples of species closely related to J. blancoi (Mao et al. 2010): J. scopulorum and J. virginiana (from Arkansas and New Mexico, respectively), were provided by Robert P. Adams (BAYLOR Adams No. 10220–10222 and No. 10933–10935). As an outgroup, one sample of Callitropsis forbesii (El Dieciseis, Baja California at 32280 0600 N, 16340 5800 W) was provided by P. Rosas and D. Gernandt from the Instituto de Biologı́a, Universidad Nacional Autónoma de México. Sampling and description of populations All eight known populations of J. blancoi and its varieties (Table 1) were sampled during 2008 and 2009. In the riparian populations, collections were made from both sides of the stream beds along a 1 km transect, allowing 20–25 m between individuals. In the subalpine populations, samples were collected all over the mountain top, allowing at least 15 m between specimens. Fresh leaves of 20–22 adult (unless otherwise specified) individuals per population were collected and stored on ice for transportation to the laboratory, and then stored at -70C. Herbarium specimens were deposited at the Herbario Nacional de México (MEXU; AMY 001-009 and 016). Conservation conditions and threats were described based on field observations and land use. Height, diameter, sex and growth form of each individual were recorded. A one-way ANOVA was performed to search for significant differences between populations in DNA isolation and genotyping DNA extraction of each sample was performed with a MINI-PREP CTAB method modified of Vázquez-Lobo (1996) and corroborated in a 1% agarose gel dyed with 0.1% ethidium bromide. The trnC-trnD cpDNA region was amplified with primers trnC and trnD (Demesure et al. 1995), in a final 123 123 N estimated number of established trees in the population; n number of sampled trees; h haplotype diversity; k number of haplotypes; BAPS group according to BAPS analyses * Differences of values followed by the same letter are not significant (P \ 0.05) for the height (a–d) and diameter (e–g), standard deviations are shown in brackets 8 0.528 (3) 19.445106, -98.574342 J. blancoi var. blancoi (viii) La Preciosita 15.8 (SD = 5.8) d 42.8 (SD = 18.9) g 200 (21) 6 7 0.627 (3) 0.706 (4) 22 (22) 90 (18) 19.762528, -100.149361 19.652639, -100.124083 22.3 (SD = 12.8) f 46.1 (SD = 17.9) g (vii) San José del Rincón 7.4 (SD = 3.6) c J. blancoi var. blancoi J. blancoi var. blancoi (vi) Presa Brockman 14.1 (SD = 3.8) d 5 4 0.524 (2) 21.351944, -103.567389 22.7 (SD = 9.9) f 5.7 (SD = 3.1) a,c J. blancoi var. blancoi (v) Rı́o Patitos 2.7 (SD = 1.1) b J. blancoi var. blancoi (iv) El Salto – J. blancoi var. huehuentensis (iii) Cerro Huehuento 50 (19) 150 (21) 23.756317, -105.378333 8.3 (SD = 6.6) e 0.524 (2) 2 3 270 (21) 24.075028, -105.739500 – 0 (1) 1 0 (1) 300 (17) 25.960110, -107.050288 0.544 (3) 220 (21) 28.374722, -108.763056 J. blancoi var. huehuentensis – 12.9 (SD = 5.4) e 4.8 (SD = 1.6) a,b J. blancoi var. mucronata (i) Yécora (ii) Cerro Mohinora – Height (m)* Taxon Population Table 1 Biological characteristics, estimates of genetic diversity and genetic structure in populations of J. blancoi N (n) Lat, long Diameter (cm)* h (k) BAPS Conserv Genet volume of 35 ll, under the following PCR conditions: (1) 5 min of initial denaturation at 80C, (2) 95C for 1 min, 58C for 4 min (30 cycles), and (3) 10 min of final extension at 66C (modified from Shaw et al. 2005). Reaction success and expected product size were confirmed on a 1.2% agarose gel dyed with 0.1% ethidium bromide. PCR products were purified and sequenced at the High-Throughput Genomics Unit (HTGU), University of Washington. Due to the region length and molecular characteristics, internal primers for sequencing were designed (pNpM and psbM54; Online Resource 2) based on J. scopulorum sequences (GenBank accession numbers EF608988, EF608989 and EF608990). Sequencing was done in both directions. A total of 160 resulting sequences were manually aligned using BioEdit Sequence Alignment Editor 7.01 (Hall 1999). Sequences of the haplotypes found are available at GenBank (accession numbers HQ651894HQ641908). Regions trnS-trnG2S (Shaw et al. 2005) and T-trnL-trnF (Taberlet et al. 1991) were also explored, but no variation was found. Genetic analyses Haplotype diversity (h) and haplotype number (k) were calculated using Arlequin 3.5.1.2 (Excoffier et al. 2007) for each population and for the whole sample. Indels of more than 1 bp and an inversion were considered as a single mutational event (Müller 2006), coded as a single gap and treated as a fifth state for all analyses except those of demographic history and neutrality tests, for which they were excluded. Only non-coding regions were used (trnCpetN, petN-psbM, psbM-trnD) for a total of 1351 bp aligned length. Genetic structure was evaluated using different approaches. First, Dest (Jost 2008) was calculated using SMOGD 1.2.5 (Crawford 2010) with 1000 bootstrap replicates. SMOGD works with diploid data, so sample sizes were doubled before running it. Second, a Bayesian spatial analysis of population structure was performed with BAPS 5.1 (Corander et al. 2008), which uses stochastic optimization to find the optimal partition. Simulations were run from K = 2 to K = 10 with 100 replicates for each K, for clustering of groups of individuals and spatial clustering of groups (Corander and Marttinen 2006). Phylogenetic relationships of haplotypes were inferred using Maximum Parsimony (MP) as implemented in PAUP* 4.0b10 (Swofford 2003), including the sequence from C. forbesii as outgroup and coding gaps as a fifth state and as a single event in the case of the indels of more than one bp. Trees were found through a branch-and-bound search with 100 replicates of branch swapping by tree bisection and reconnection (TBR). Branch support values were estimated Conserv Genet Table 2 Geographic distance (lower diagonal) and genetic differentiation (Dest, upper diagonal) between J. blancoi populations Yécora Yécora Cerro Mohinora Cerro Huehuento El Salto Rı́o Patitos Presa Brockman San José Rincón La Preciosita 1 1 1 0.296 1 1 1 0.840 0.833 1 1 1 1 0.360 1 1 1 1 1 1 1 1 0.846 0.519 0.674 0.316 0.757 Cerro Mohinora • Cerro Huehuento • • El Salto j • h Rı́o Patitos j j • • Presa Brockman m j j j • San José del Rincón La Preciosita m m j m j m j m • j h • 0.504 • Categories for geographic distance: filled triangle larger than 900 km; filled square from 400 to 899 km; filled circle from 150 to 399 km; open square from 14 to 50 km. There are no populations separated by 51–149 km from 1000 replicates of bootstrap. TCS version 1.21 (Clement et al. 2000) was used to infer haplotype networks using statistical parsimony (Templeton et al. 1992) with a confidence limit of 95%. In order to test for isolation by distance, Mantel tests were performed between the Dest and geographical distance matrices with 10,000 permutations in Arlequin 3.5.1.2 (Excoffier et al. 2007). A distance matrix was generated using the logarithm of geographic distance (Rousset 1997), calculated with Geographic Distance Matrix Generator 1.2.3 (Ersts 2009). Possible population expansion was explored with Tajima’s D-test (1989) and Fu and Li’s Fs test (1993). Both analyses were performed in Arlequin 3.5.1.2 (Excoffier et al. 2007) using only the nucleotide differences, so the sample was reduced to three haplotypes (H1, H5 and H7, Online Resource 1). morphology. Differences in growth form and quantitative traits within populations of the type variety can be considered as evidence to reject the hypotheses of recent ecological exchangeability. Exchangeability Results Categories of population distinctiveness based on rejection or failure to reject the null hypotheses (H0) of genetic and ecological exchangeability were assessed. The null hypothesis of genetic exchangeability is rejected when there is evidence of restricted gene flow between populations and the ecological exchangeability is rejected when there is evidence of population differentiation (Crandall et al. 2000). Historical genetic exchangeability can be rejected if evidence from isolation is found according to Dest, BAPS, and haplotype networks. Recent genetic exchangeability is rejected when gene flow is determined to be not likely due to geographic isolation, geographic distance, and reproductive biology. Historical ecological exchangeability is rejected with the presence of varieties within the species as evidence of differences in habitat, environmental conditions, and Juniperus blancoi populations and rarity Conservation units Conservation units were defined following a qualitative approach. Fraser and Bernatchez’s (2001) practical criteria for defining ESUs and Green’s (2005) recommendations for recognizing populations in need of protection were used. Starting with all the populations grouped according to BAPS, they were re-grouped considering the variety, habitat, growth form, morphological differences, and evolutionary history that comprise the ecological and genetic distinctiveness of each population. The species has eight known populations and three recognized varieties (Table 1). J. blancoi var. blancoi grows exclusively in stream margins as trees or shrubs from 2 to 25 m in height (Adams and Zanoni 1979; Zamudio and Carranza 1994; Pérez-de la Rosa and Carrillo-Reyes 2003). J. blancoi var. mucronata shows foliar differences with respect to other varieties (Adams 2000). It is a riparian tree or shrub of shorter height and constitutes the northernmost population of the species. J. blancoi var. huehuentensis is a subalpine, prostrate shrub form that grows on bedrock and very thin soil on the upper slopes of some of the highest peaks of the Sierra Madre Occidental (Adams et al. 2006). All populations were sampled. Five of the locations were discovered within the last 10 years, including La 123 Conserv Genet Table 3 Main environmental and morphological characteristics of J. blancoi’s CUs CU Taxon Populationsa Habitat Elevation (masl) Growth form I J. blancoi var. mucronata i Riparian, rocky soil 1,300 Shrub–tree II J. blancoi var. huehuentensis ii and iii Subalpine, mountain summit, rocky soil 3,150–3,200 Postrate shrub III J. blancoi var. blancoi iv Riparian, deep & partially rocky soil 2,500 Shrub–tree IV J. blancoi var. blancoi v Riparian, rocky soil 1,700 Shrub–tree V J. blancoi var. blancoi vi–viii Riparian, deep soil 2,700–2,800 Tree a Numerals correspond to populations as in Table 1 Preciosita and Cerro Mohinora (collected in 2008 and 2009, respectively), for which this is their first public report. The species grows from 1,350 to 3,250 masl, but such extremes correspond to J. blancoi var. mucronata and J. blancoi var. huehuentensis, respectively, while the populations of the type variety were found around 2,700 masl (Table 3). There was phenotypic variation in tree diameter and height among some populations. Considering the maximum values per population, J. blancoi var. blancoi height varied from 5 to 26 m and from 28 to 82 cm in diameter, with some populations showing significant differences (Table 1). It was not possible to measure J. blancoi var. huehuentensis height and diameter due to its form of growth (prostrate shrubs). With regard to sex, each population had a 1:1 ratio of males:females. Genetic variation and structure A total of 1555 bp (aligned length) cpDNA were sequenced, 202 bp of these correspond to the petN (90 bp) and psbM (112 bp) genes, while the rest correspond to the intergenic spacers (Online Resource 1). In such non-coding regions, three different kinds of mutations were found: SNPs, indels, and inversions, which generated 12 polymorphic sites and 10 different haplotypes in J. blancoi (Online Resource 1). The most frequent haplotypes were H5 (20%), H7 (19.37%), and H1 (17.5%). Haplotype diversity was high (hT = 0.863), but the diversity in each population differed across the range (Table 1). The TMVB populations showed more diversity than the ones from SMO (Fig. 2a). The population with the highest diversity was Presa Brockman, followed by San José del Rincón (he = 0.706 and 0.627 respectively), while Yécora and Cerro Huehuento had a fixed haplotype (H5 and H7, respectively; Table 1). No haplotype is present in all populations, 50% of the sampled haplotypes are private to a given population, and half the populations have at least one private haplotype (Fig. 2a). While some populations are not genetically diverse, differences in haplotype composition were found all over 123 the range (Fig. 2a), which increases the total diversity and differentiation values. Most pairwise Dest estimates were large even between geographically close populations (Table 2). For example, San José del Rincón and Presa Brockman, which are 14 km from each other, had a Dest = 0.316. There is a strong total species differentiation, as shown by Dest = 0.866. A test of clustering of groups of individuals performed with BAPS showed eight as the number of best partitions, with a Log (marginal likelihood) = -1315.0915. Spatial clustering also showed eight with a slightly larger Log (marginal likelihood) = -1330.5763. Two different haplotypes were found in J. scopulorum and two in J. virginiana out of the three samples provided from each species. Indels, SNPs and microsatellites were found (272–286 and 279–298 regions shown in Online Resource 1). MP tree (Fig. 2c) shows that J. blancoi’s haplotypes are paraphyletic. The node of the H5 haplotype is not resolved. The phylogenetic tree illustrates that the geographic distribution of the haplotypes is congruent with the conformed clades: H1 and H5 are in both SMO and TMVB; while H6, H7, H9 and H10 are only in SMO and H2, H3, H4 and H8 are only in TMVB. The Mantel test showed a significant correlation between Dest and geographic distance (r2 = 0.629, P = 0.003). Tajima’s D-test and Fu and Li’s Fs-test were statistically non significant (D = 0.798, P [ 0.1 and F = 1.125, P [ 0.1, respectively) so the sudden expansion hypothesis was rejected. Exchangeability and conservation units The two hypotheses of genetic and ecological exchangeability were rejected. As discussed below, evidence to state this are the distribution of the species and form of rarity; its marked genetic structure; high differentiation; morphological differences (both in the form of taxonomic varieties and size and growth form variance); range of environmental conditions; evolutionary history, and dispersability. This corresponds to Crandall et al. (2000) Case 1 of distinctiveness. Five CUs are proposed (Table 3). Conserv Genet Discussion Genetic exchangeability Juniperus blancoi form of rarity Genetic exchangeability is rejected when there is evidence of restricted gene flow between populations (Crandall et al. 2000). Due to the nature of the data it was not possible to measure gene flow or to estimate the precise time to the most recent common ancestor (tMRCA). However, the hypothesis of historical genetic exchangeability was rejected because the high Dest values, 8 BAPS groups, the presence of private haplotypes, and the fact that missing haplotypes are needed to connect the haplotype network suggest that at least in the SMO populations, a fragmentation process could have occurred in the past, followed by restricted gene flow between populations. The hypothesis of recent genetic exchangeability was also rejected because the species has a geographic distribution characterized by small populations separated by long distances, along with specific habitat preferences and possibly a low dispersability, all of which suggest restricted gene flow between the current populations. Strong fragmentation could be suggested by the high pairwise Dest values (Table 2) found among populations. Such differentiation is also the explanation for J. blancoi’s high variation: even if many populations are not highly diverse, differences in the haplotype composition and the presence of private haplotypes increase total diversity. Haplotype composition and diversity is such that, according to BAPS, individuals cluster together in eight different groups that correspond exactly to each population (Table 1); the same result was obtained following the spatial clustering of groups. Such structure could be a consequence of fragmentation processes, an idea also suggested by the missing haplotypes (Fig. 2b) between H5, H1 and H7 (although they could also be present in existing but not yet described populations, see below). Palaeontological evidence shows that neartic plant genera (as Juniperus is considered to be) entered Mexico from the north during the late Cenozoic and became very well represented during the Pliocene (Graham 1993). Afterward, it seems that some species could have undertaken phases of isolation and recontact along mountain corridors (e.g. JaramilloCorrea et al. 2008; Gugger et al. 2011) as a consequence of altitudinal changes in their distribution during the Pleistocene’s glacial cycles (Lozano-Garcı́a et al. 2005; Ortega-Rosas et al. 2008). It is possible that J. blancoi was similarly affected by events in the past, with population fragmentation and isolation as one of the consequences. Isolation could have been produced by physical distance and/or ecological differences that could be maintained up to the present. The Mantel test showed a significant correlation between differentiation and distance, which is not surprising in populations separated by up to 900 km (Table 2). In junipers, chloroplast inheritance is paternal Juniperus blancoi populations are small and scattered across its distribution range. Riparian populations follow waterways for only about 1–3 km and then disappear even if the forest is not perturbed. In the populations showing the least human intervention (La Preciosita, Cerro Mohinora, and Cerro Huehuento) no more than 300 adult individuals were found in the entire area (approximately 2.2, 3, and 4 ha, respectively). The species area of occupancy is not greater than 10 km2, but it is locally dominant. In a previous study in La Preciosita (Nepomuceno-Martı́nez et al. 2007) 95% of the trees with a diameter C1 m sampled at each side of the stream were J. blancoi. Gadow’s mixture index (del Rı́o et al. 2003) was close to zero (DM = 0.062), which means that it is an almost pure mass of the species. Although there is no similar analysis for the other populations, their characteristics are very similar: few scattered individuals of other tree species are found, if any, at each locality. Thus, J. blancoi distribution is wide yet restricted, and it is dominant in its very particular niche. This is a specific form of rarity according to Rabinowitz’s (1981) matrix and, most probably (see discussion below), this species could owe its distribution to environmental changes in the Pleistocene, which implies that it could be labeled as a long time and narrowly distributed rare species according to the classification of Fiedler and Ahouse (1992). These authors describe taxa in this category as those distributed over a small area (or highly clumped) for a long period of time, but also those (like Sequoia sempervirens) that historically were not rare, but are so today. There are many other rare conifers in Mexico (Farjon and Styles 1997; Adams 2008) that also could have had broader distributions during cold periods in the past. However, little is known about how Quaternary climate fluctuations affected cold-adapted species in tropical and subtropical latitudes, so it is difficult to assign them to accepted categories of refugia (Stewart et al. 2010). It is a common belief that rare species should have little genetic variation due to their small population sizes, but various studies have shown that rare species have similar or greater genetic variation than widely distributed species (Gitzendanner and Soltis 2000; Molina-Freaner et al. 2001; Newton et al. 2002; Kettle et al. 2007; Rosas Escobar et al. 2011). J. blancoi has high genetic variation (hT = 0.863), that is greater than that reported in a similar study with cpDNA sequences in the Chinese J. przewalskii (hT = 0.568, Zhang et al. 2005), of wider distribution. This again highlights the importance of considering the historic processes behind rarity. 123 Conserv Genet (Neale et al. 1989; Mogensen 1996) and pollen is distributed by wind. Due to the smaller effective population size of cpDNA compared with nDNA, lower differentiation values could be expected. However, pollen movement is not so easy; in some pines a single grain does not travel more than 300 m (Lian et al. 2001). It is true that in other pines effective pollen flow (up to 4.4%) was detected from a population located 100 km away (Robledo-Arnuncio 2011), however that was a considerably large population. Mentioning this is important because pollination success depends on finding a fertile ovuliferous cone, so it is related to population density. Due to small population sizes and large distances between localities, pollen flow between current populations of J. blancoi seems unlikely, especially for population pairs separated by more than 150 km (Table 2). Similarly, it is likely that seed dispersion would not be high because seeds are not winged. However, it is known that the fleshy female cone typical of the genus serves as food for some birds and small mammals (Santos et al. 1999). There is no specific study for this species, but young plants are commonly close to pocket gopher burrows (AMY personal observation); these animals are characterized by low mobility and marked genetic differentiation and structure (Patton and Yang 1977; Hafner et al. 1987), so it is likely that they would not disperse the seeds far away. Ecological exchangeability We consider the marked genetic structure, the presence of different clades in the haplotype network (Fig. 2b), and the existence of different varieties as evidence to reject the hypothesis of historical ecological exchangeability. As discussed below, this differentiation could be related to hydrological and climatic changes in the study area, leading some of the species’ populations to isolation under different environmental conditions, which could be related to the morphological differentiation of its described varieties. However, there is also variation within J. blancoi var. blancoi populations (Table 1) that could indicate that these localities are under different environmental regimes as well. In the last 3.5 Myr, orographic and climatic changes occurred in SMO and TMVB. The direction of the streams and the basin limits varied until reaching the present conformation (de Cserna and Álvarez 1995), and the conditions dried up and became hotter at the end of the Pleistocene (McAuliffe and Van Devender 1998; Herbert et al. 2001; Caballero and Guerrero 1998; Metcalfe et al. 2000). Geographic barriers and changes in the connectivity of streams could have isolated J. blancoi populations. For example, Rı́o Patitos (SMO), Presa Brockman and San José 123 del Rincón (TMVB) populations are found in the LermaSantiago Basin (Fig. 2a), which used to include a larger system of lakes and streams that dried up during different events in the Pleistocene (West 1964). At the same time, suitable habitat could have decreased due to changes in climatic conditions. Oaks and coniferous forests from SMO and TMVB are currently separated by dry forest and xerophytic shrub (principally), but according to paleoclimatic studies such areas could have had coniferous forest including junipers in the past (Lozano-Garcı́a et al. 2005; Vazquez and Gaston 2004). Populations recognized as varieties could have been isolated under specific environmental conditions. Particularly on mountain tops where J. blancoi var. huehuentensis (Fig. 1b; Table 3) inhabits, local adaptation could have developed. Morphological differentiation (prostrate shrub, leaves protecting seed cones) possibly associated with a colder mountaintop without running water (Adams et al. 2006) could be considered evidence of this. Regarding the variation within J. blancoi var. blancoi, TMVB individuals are large trees, up to 20 m high, that grow in deep soils next to small water streams (Fig. 1a), while the SMO population are significantly smaller trees or even shrubs growing either on a very rocky river side (Rı́o Patitos) or in deep soil but with the rocky formations (El Salto) of a small water stream. The San José del Rincón population is significantly smaller than the other two TMVB populations; however, we believe this to be an effect of human intervention. This population is endangered in terms of conservation, so very young trees were collected in order to have enough samples. This lowered the height and diameter means, but some large trees, up to 15 m high, were also found. Variation in height, diameter and growth form could be explained by phenotypic plasticity; however, it has been proven that they also have a genetic component (Bradshaw 1965; Kaya et al. 1999; Sultan 2000, 2003) related to adaptations to local environmental conditions, such as differences in temperature and habitat (Table 3). Provenance tests showing differences in drought and frost tolerance between other conifer genera can be found elsewhere (e.g. Oleksyn et al. 1998; Blada and Popescu 2007; Laguna et al. 2008; Reich and Oleksyn 2008). For the Mexican region, there are some studies showing smaller, twisted pines in drier and colder populations in the north and larger and straight trunk trees in warmer and more humid populations in the south (Viveros-Viveros et al. 2005; Saenz-Romero et al. 2006; Saenz-Romero and Tapia-Olivares 2008). There is no common garden study for J. blancoi, but likely it would correspond to the described differences among populations. So even if the height, diameter and growth form are not characteristics defining the species, we consider them to be evidence to Conserv Genet reject the hypotheses of recent ecological exchangeability in J. blancoi. Conservation units As stated above, there is genetic and ecological evidence to suggest that J. blancoi populations have restricted gene flow and have begun to show phenotypic differences possibly related to differences in environmental conditions and long-term isolation. This is congruent with Fraser and Bernatchez’s (2001) definition of ESU as a lineage demonstrating highly restricted gene flow from other such lineages within the higher organizational level of the species. The rejection of ecological and genetic exchangeability, both historical and recent, as proposed for J. blancoi, is Crandall et al.’s (2000) Case 1 of distinctiveness. This means that populations, or population groups within the species, should be considered as separate units. Five CUs are proposed (Table 3). CU I consists of the Yécora population (Fig. 1c), recognized as J. blancoi var. mucronata. It is fixed for the haplotype H5, which could be the ancestral. This population could be considered a remnant of the older and more northern distribution of the species. It grows in a small temperate flora region within arid vegetation. Although it is in a marked glen of difficult access both for humans and livestock, it is a small population in the middle of agricultural land, so it has a certain level of threat. CU II would correspond to J. blancoi var. huehuentensis. Although these populations share a haplotype (H7) with a population of J. blancoi var. blancoi (El Salto), we considered them as a separate unit because the individuals of the subalpine variety are morphologically distinctive shrubs that grow on rocky soil above 3,150 masl (Fig. 1b). As discussed before, this makes them non-ecologically exchangeable with El Salto, and possibly implies restricted pollen flow between populations. There is no human pressure on these high altitude areas, which are within a Natural Protected Area and a community forest management unit, so climate change seems to be the only threat for them. CU III would correspond to the El Salto, as it seems to be non-exchangeable with the other populations of the same variety: it is in a different clade than them and has its own private haplotype (Fig. 2); the geographically closest population (Rı́o Patitos) is *200 km away, in a different basin (Fig. 2a) and a much more rocky environment (Table 3); the other populations, while more similar in habitat, are farther away in the TMVB. The El Salto population is mainly in the middle of pasturelands, where soil is eroded, few trees remain and cows do not allow the establishment of new plants. CU IV, the Rı́o Patitos population, also corresponds to the type variety. It represents, both geographically and historically, the intermediate population between SMO and TMVB: physiographically it grows in the former, hydrographically in the latter, and it shows a genetic composition (haplotypes H1 and H5) related to both (Fig. 2). Its habitat and growth form are similar to those of the Yécora population; however, it does not share the morphological differences in the leaves. From the conservation perspective it is also in a similar situation to Yécora. Finally, CU V corresponds to the TMVB distribution of J. blancoi var. blancoi (Fig. 1a). The highest genetic variation is found here; haplotypes are in a monophyletic clade (Fig. 2c) and are restricted to the area except for H1, which links them to the SMO populations. This CU could be the most recent lineage of the species, and seems to be less historically fragmented than the SMO populations. However, the Presa Brockman and San José del Rincón populations, especially the former, are highly threatened because they are in the middle of agricultural land. In San José del Rincón there are fewer than 25 individuals. La Preciosita is safe in a community forest reserve, so if necessary junipers from here could be used to help to rescue the other TMVB populations. As regards the finding of recently discovered populations, if new localities appear, they would probably also be small and restricted to a specific area; otherwise they would had been described already. It is difficult to tell what their genetic variation might be, but it is possible that they could contain some of the missing haplotypes (Fig. 2b) or present some of the current main haplotypes along with new private ones, as was the case in La Preciosita and Cerro Mohinora. Thus, finding more small populations will imply that the species will continue to be considered rare, with a fragmented distribution and a marked genetic structure. This species is a good example of how population units could show different conservation status (and need different conservation strategies) than if considering the species as a single entity. This is not only because of their evolutionary background, but as highlighted by Green (2005), also for its present conservation conditions. Although not differentiated as species, we propose that these CUs represent non-exchangeable evolutionary units whose conservation must be considered independently. Conclusions The form of rarity of J. blancoi must be related to history. Its wide distribution in isolated small populations that show an overall high genetic diversity and large differentiation could be a consequence of fragmentation and isolation of a wider distribution; such fragmentation could be related to Quaternary climate fluctuations. Other subtropical and tropical conifers characterized by presenting rare distributions and morphological variation (as, for example, many Mexican endemic pines and junipers do; Farjon and Styles 123 Conserv Genet 1997; Adams 2008) could also show this behavior (MolinaFreaner et al. 2001; Newton et al. 2002; Cuenca et al. 2003). Thus, showing that J. blancoi’s evolutionary history has led to ecological and genetic distinctiveness within its populations is important not only for its conservation, but also as part of the necessary steps to increase the evolutionary understanding of biodiversity in one of the hot spots of conifer species. J. blancoi conservation should be taken not as a single entity, but as the above-mentioned five CUs representing lineages that are historically, ecologically and genetically distinct from each other. Conserving these small populations has consequences in conserving a complex evolutionary history of non-exchangeable populations. As a species, J. blancoi must be listed in the Mexican NOM-059-SEMARNAT-2010 list of endangered species, and its category in the IUCN Red List should be reviewed. The ecological and genetic distinctiveness of its populations should be considered when doing so. Acknowledgments We thank La Preciosita and Cerro Huehuento’s communities for conserving the species populations and helping us during the sampling; Pilar de la Garza, Felipe Nepamuceno and INIFAP for initial studies; Socórro González, David Ramı́rez, CIDIIR-Dgo. Francisco Molina, and Jose F. 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