Spines and ribs of Pilosocereus arrabidae (Lem.) Byles & G.D. Rowley and allies (Cactaceae): Ecologic or genetic traits?

Flora 214 (2015) 44–49

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Spines and ribs of Pilosocereus arrabidae (Lem.) Byles & G.D. Rowley and allies (Cactaceae): Ecologic or genetic traits? Marcelo O.T. Menezes a,d,∗ , Nigel P. Taylor b , Daniela C. Zappi c , Maria Iracema B. Loiola d a

Instituto Federal do Ceará, Departamento de Ensino Médio e Licenciatura, Av. Treze de Maio 2081, Fortaleza,CE 60040–215, Brazil Singapore Botanic Gardens, National Parks Board, 1 Cluny Road, 259569, Singapore Conservation Science Team, Royal Botanic Gardens, Kew. TW9 3AB, Richmond, Surrey, United Kingdom d Universidade Federal do Ceará, Departamento de Biologia, Av. Humberto Monte S/N, Campus do Pici, Bloco 906. Fortaleza, Ceará, 60455–970, Brazil b c

a r t i c l e

i n f o

Article history: Received 15 April 2015 Received in revised form 25 May 2015 Accepted 29 May 2015 Available online 1 June 2015 Keywords: Rainfall Morphometry Inheritance Taxonomy Genetic distance Columnar cacti

a b s t r a c t Amongst the more outstanding features of columnar cacti, their ribs and spines may vary greatly between and within species. With the implicit assumption that morphometric traits of spines and ribs are inherited, these features have been historically used for taxonomic purposes in Cactaceae. However, some studies show that environmental variables may influence morphology of cacti and thus, some traits are not solely genetically determined. We aim to test the influence of both genetic and environmental factors upon spine and rib patterns. We tested the correlation between environmental, genetic, and morphological data on three taxa of Pilosocereus from eastern Brazil (from the Pilosocereus arrabidae group). We evaluated the influence of those factors in three traits: the length of the longest spine in the areole, the number of spines per areole and number of ribs. The number of ribs exhibited a significant positive relationship with genetic distance. The length of the longest spine is positively correlated with rainfall, but the number of spines per areole is negatively correlated. Negative relationships were also found between latitude and number of ribs, as well as between latitude and the number of spines per areole. Our results advise towards cautious use of these traits in cactus taxonomy. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Areoles, ribs, and spines – outstanding morphological features of columnar cacti – are part of the adaptive strategy of these plants to arid and semiarid environments. Members of the Subfamily Cactoideae (the most diverse and derived of the family) bear no functional leaves. Thus, photosynthesis is performed exclusively by the stem, which is adapted to perform typical leaf tissue functions and has a similar morphological organization (Sajeva and Mauseth, 1991). The areoles are lateral shoots with no internodes, from which new branches, spines, and flowers arise (Rowley, 2003). In columnar cacti these structures are often arranged in protruding vertical series called ribs, which produce a folded stem surface. This shape affects the shoot’s properties, such as surface-to-volume ratio, strength, flexibility, and self-shading (Mauseth, 2006). According to Mauseth (2006) and Perez-Harguindeguy et al. (2013), the rib traits (mainly number and height) may be functional features that

∗ Corresponding author at: Instituto Federal do Ceará, Departamento de Ensino Médio e Licenciaturas, Av. Treze de Maio 2081, Fortaleza, Ceará, 60040–531, Brazil. E-mail address: [email protected] (M.O.T. Menezes). http://dx.doi.org/10.1016/j.flora.2015.05.008 0367-2530/© 2015 Elsevier GmbH. All rights reserved.

respond to solar radiation just as leaf area in leaf-bearing plants. Spines, which are modified leaves (Boke, 1944, 1980), are commonly found in most representatives of Cactaceae. Despite being usually considered a defense against herbivores and other environmental factors such as fire (Perez-Harguindeguy et al., 2013), spines can assume many functions related to physiology, reproduction, and ecological interactions (Mauseth, 2006; Loik, 2008; Drezner, 2011; Guerrero et al., 2012). Rib and spine traits may vary greatly between tribes, genera, species, populations, and even within populations (Mauseth, 2006). Due to the intense variation between different taxonomic groups, morphological traits such as position and number of spines per areole, spine length and number of ribs have been widely used to delimit and identify genera, species or subspecies (e.g. Hunt et al., 2006; Schmalzel et al., 2004). An implicit assumption that these traits are innate (or at least widely influenced by genetics) underlies their use in taxonomy. Although, there is some evidence that they are inheritable (Mihalte and Sestras, 2012), the factors involved with spine and rib morphogenesis are still poorly known (Mauseth, 2006). Furthermore, it is also known that spine and rib traits can be influenced by the environment, including growth conditions (e.g. Arellano and Casas, 2003; Casas et al., 1999;

M.O.T. Menezes et al. / Flora 214 (2015) 44–49

Schmalzel et al., 2004; Peharec et al., 2010) and ecological interactions (Pérez–Harguindeguy et al., 2013). Nobel (1983), Loik (2008), and Drezner (2011) found that the spine coverage directly affects the downward solar radiation on the stem. Thus, dense spine coverage and high number of ribs may provide shelter against excessive radiation, which can disturb photosynthetic efficiency (Loik, 2008) or cause overheating (Nobel, 1983); Drezner (2011) also found that ribs can help to promote heat loss. In an opposite situation, in low radiation environments, dense spine coverage can block the already scarce radiation (Loik, 2008). Consequently, each cactus must evolve its spine coverage pattern in order to maximize its photosynthetic efficiency within its own habitat. In some cases, stems from the same individuals/populations can present remarkably different spination patterns in response to different degrees of sunlight exposure. For example, Majure (2007), studying Opuntia pusilla (Haw.) Haw., found that stems grown in the full sunlight presented dense spine coverage, while shaded stems grew with few or no spines. Considering the importance of vegetative morphological traits to the taxonomy of the family, we aim to investigate the role of genetic and environmental variables in determining morphometric traits of cacti grown in natural conditions. We studied spine length, number of spines per areole, and the number of ribs in Pilosocereus arrabidae (Lem.) Byles & G.D. Rowley and two subspecies of Pilosocereus catingicola (Gürke) Byles & G.D. Rowley (the “Arrabidae group” sensu Zappi, 1994) in relation to genetic and environmental data. Our predictions are: (1) if these traits are genetically determined, the pair-wise trait difference between two plants would be proportional to their pair-wise genetic distance; or (2) if environmental factors are more important than genetics, then there would be a correlation between the trait’s measurements and environmental variables.

2. Material and methods Numeric data on the longest spine’s length, number of spines per areole and number of ribs were obtained from 35 dry specimens from the EAC herbarium (Table 1). We selected 20–30 areoles from each plant to count the number of spines and measure the longest spine (with a simple caliper rule). These measurements were used to calculate a mean for each specimen. The number of ribs was counted from the cross section of each exsiccatum. Whenever the specimen had cross sections with different numbers of ribs, we used a mean value. Since, spines are often damaged during the drying process, we did not take measurements from areoles with broken spines nor from areoles with missing spines, which are both noticeable for this group of species. Reproductive areoles were ignored too, because they are often more or less modified in relation to the sterile ones. We used local mean annual rainfall (mm/year) as a measure of water availability for each specimen analyzed. Precipitation data were obtained from http://climate-data.org (AmbiWeb, Gernsbach, Germany). Latitude, recorded with GPS during the collection of each sample, was used as an indirect measure of the downward solar shortwave radiation (as it decreases polewards; Hatzianastassiou et al., 2005). Genetic sequences were obtained from two non-coding intergenic spacers of chloroplast DNA (trnT-trnL and trnS-trnG). These markers were successfully used for Pilosocereus and are known to exhibit variation within species (Bonatelli et al., 2013, 2014). DNA was extracted from tissue samples using the traditional CTAB procedure. The DNA sequences were amplified with GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA). PCR protocol followed the standard application suggested by the manufacturer, using annealing temperatures described by Bonatelli et al. (2013).

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The PCR products were purified with EZ-10 Spin Column Purification Kit (Bio Basic Inc., Markham, Ontario, Canada) and sequenced with ABI 3730 XL DNA Analyzer (Applied Biosystems, Foster City, California, USA). The tips of the sequences and the homonucleotide repeats (one from trnT-trnL and two from trnS-trnG) were removed from the analyses due to ambiguity and uncertain homology. Sequences of each marker were then aligned using Muscle (Edgar, 2004). Gaps and inversions were manually coded as presence (1)/absence (0) information at the end of the respective sequence. 3. Calculation To test our hypotheses, environmental, genetic, and morphological data were tested for correlation and linear regression. First, the data of each variable were tested for normality with a Shapiro–Wilk test. Since most of the variables were not normal, we used the nonparametric Spearman’s rank correlation coefficient (rho). Numeric relationships with rho lower than 0.4 were considered weak; those with absolute values of rho higher than 0.6 were considered strong. All statistic tests were done in R (R Core Team, 2014). We tested the correlation between the mean value of each morphological trait (longest spine’s length, number of spines per areole and number of ribs) and each environmental factor (mean annual rainfall and latitude). For these tests, we used a subset of 31 samples of P. arrabidae, P. catingicola subsp. catingicola and P. catingicola subsp. salvadorensis (our focal species group). For the tests on genetic distance, we used a subset of 29 samples (one DNA sample of P. arrabidae and four of P. catingicola subsp. salvadorensis failed to amplify in at least one marker, even after repeated attempts). We created 406 pairs of genetic and morphological distances from these samples. The pair-wise genetic distance between two samples was measured as the number of mutations (substitutions, inversions, and gaps) in which their sequences differed. This count was made by means of a haplotype network based on the median-joining network algorithm (Bandelt et al., 1999), implemented in Network v4.6 (Fluxus Technology Ltd., Clare, Suffolk, England). These values of genetic distance were tested for correlation with the respective pair-wise difference between the mean value of each morphological trait. 4. Results The whole alignment (including the gaps) was 1414 bp long. After the removal (and coding) of the gaps, this became 1245 bp long. We found eight different sequences (haplotypes), with 14 polymorphic sites, three gaps and one inversion (Table 2). Hereafter, the haplotypes are called from H1 to H8. Only H1 was shared by P. arrabidae and both subspecies of P. catingicola. The others were exclusive to their respective taxa (Table 1). The genetic distance between populations or taxa varied from zero (in the case of the shared haplotype) to 11 mutations (Table 2). Genetic distance was lower among taxa of the Arrabidae group. The mean length of the longest spine varied from 8.25 to 36.05 mm; the mean number of spines per areole, from 6.5 to 22.4; and the mean number of ribs, from 4.5 to 16. Latitude varied from 3.38 to 22.92 decimal degrees and mean annual rainfall from 504 to 1614 mm/year (Table 1). A huge variability was found in the traits of each taxon of the Arrabidae group. P. catingicola subsp. salvadorensis exhibited the greatest variability. This taxon was also the most widespread and found in wider ranges of latitude and precipitation (see Table 1). Only the number of ribs exhibited significant relationship with genetic distance, which was positive and moderate; the other traits did not (Table 3). The linear model for this relationship was (1) R = 2.6365 + (0.5783 × G), where “R” is the

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Table 1 Location, coordinates (decimal degrees), mean rainfall, voucher, mean traits, and haplotypes of the specimens used in the study. Locality

Coordinates (S, W)

P. arrabidae

Mucuri, Bahia Santa Cruz de Cabrália, Bahia Guarapari, Espírito Santo Araruama, Rio de Janeiro Cabo Frio, Rio de Janeiro Macaé, Rio de Janeiro Rio das Ostras, Rio de Janeiro Caraíbas, Bahia Ipirá, Bahia Itiúba, Bahia Jacobina, Bahia Manoel Vitorino, Bahia Morro do Chapéu, Bahia Nova Fátima, Bahia Rafael Jambeiro, Bahia Tanquinho, Bahia Alto Santo, Ceará Aquiraz, Ceará Aracati, Ceará Cascavel, Ceará Caucaia, Ceará Caucaia, Ceará Russas, Ceará Russas, Ceará Uruoca, Ceará Sertânia, Pernambuco Sertânia, Pernambuco Ac¸u, Rio Grande do Norte Itaú, Rio Grande do Norte Feira Nova, Sergipe Gararu, Sergipe Sobral, Ceará Vic¸osa do Ceará, Ceará Vitória da Conquista, Bahia Armac¸ão dos Búzios, Rio de Janeiro

18.07, 39.53 16.31, 39.02 20.61, 40.42 22.91, 42.37 22.72, 41.99 22.34, 41.75 22.48, 41.90 14.46, 41.11 12.29, 39.88 10.67, 39.81 11.07, 40.75 14.08, 40.21 11.52, 41.26 11.76, 39.46 12.56, 39.49 11.96, 39.15 5.52, 38.43 3.85, 38.38 4.69, 37.59 4.03, 38.23 3.70, 38.63 3.83, 38.78 5.05, 38.00 5.05, 38.00 3.38, 40.70 8.33, 37.27 8.13, 37.26 5.55, 37.06 5.81, 37.94 10.23, 37.36 10.01, 37.11 3.84, 40.03 3.604, 41.22 14.79, 40.97 22.78, 41.91

P. catingicola subsp. catingicola

P. catingicola subsp. salvadornsis

P. chrysostele P. pachycladus subsp. pernambucoensis P. pentaedrophorus subsp. robustus P. ulei

Mean annual rainfall (mm/year) 1583 1614 1087 993 843 1126 1056 671 676 671 831 703 691 504 647 812 784 1374 1024 1248 1326 1326 824 824 1066 566 566 645 759 800 629 808 1258 717 914

Voucher

Mean length of the longest spine (mm)

Mean number of spines

Menezes, 372 Menezes, 373 Menezes, 371 Menezes, 360 Menezes, 363 Menezes, 365 Menezes, 364 Menezes, 311 Menezes, 314 Menezes, 323 Menezes, 320 Menezes, 308 Menezes, 316; 319 Menezes, 325 Menezes, 305 Menezes, 326 Menezes, 201 Menezes, 152; 154 Menezes, 155 Menezes, 150 Menezes, 148 Menezes, 160 Menezes, 205 Menezes, 206 Menezes, 226 Menezes, 350 Menezes, 351 Menezes, 301 Menezes, 355 Menezes, 342 Menezes, 344 Menezes, 164 Menezes, 243 Menezes, 310 Menezes, 362

24.75 17.75 18.70 17.05 34.85 27.35 27.55 24.35 17.80 18.60 15.05 10.15 11.00 11.60 15.55 25.25 16.75 27.85 12.90 18.88 19.95 21.85 33.25 22.55 15.10 16.65 10.90 10.00 14.25 12.90 8.25 16.00 16.40 12.00 12.20

8.55 9.10 6.50 9.25 7.45 8.05 9.70 12.90 17.05 15.90 18.40 11.67 16.00 20.40 12.35 13.50 16.70 14.23 15.50 14.78 14.25 16.40 16.75 19.85 14.90 18.75 21.90 18.45 14.25 22.40 16.15 22.00 16.00 12.20 9.95

Mean number of ribs

6 6 6.5 6 4.5 5 6 5 6 5 5 5 5.5 7 6 6 10 9 11 – 9 9 8 8 10 16 15 8 9 8 10 25 13 10.5 8

Haplotype

H1 H1 H1 H1 H2 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H3 H3 H3 H3 H3 H3 H3 H3 H4 H3 H3 H3 H3 H1 H1 H5 H6 H7 H8

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Taxon

47

pair-wise difference in the number of ribs and “G” is the pairwise genetic distance. Two traits were significantly correlated with rainfall: the length of the longest spine and the number of spines per areole. In both cases, correlation was moderate. However, they behaved differently, with positive and negative relationships, respectively (Table 3). The linear models for these relationships are: (2) L = 10.4953 + (0.0089 × P), where “L” is spine length and “P” is the precipitation (mean annual rainfall); and (3) N = 21.2756 − (0.0073 × P), where “N” is the number of spines per areole. No significant relationship was found between rainfall and the number of ribs. The number of spines per areole and the number of ribs also exhibited negative relationships with latitude; spine length did not. These relationships were both negative, but moderate and strong, respectively (Table 3). The linear models for these relationships are: (4) N = 19.7805 − (0.4698 × D), where “D” is the latitude in decimal degrees, and (5) R = 10.6598 − (0.2622 × D), where “R” is the number of ribs.

– – – – – – – 0

H8

– – – – – – 0 10

H7

– – – – – 0 5 5

H6

– – – – 0 3 2 8

H5

– – – 0 9 6 11 8

H4

– – 0 4 9 6 11 8

H3

– 0 4 4 9 6 11 8

H2

0 . . . 1 . 1 .

0 3 1 3 8 5 10 7

H1

a

b

Inversions. Gaps.

A G . G G G G G

0 . 1 . . . . .

C . . . . . . T

T . . . C . C .

C . . . T T T A

G . . . . . A .

C . . . G G G G

G . . . A . A .

A . . C . . . .

G A . . . . . .

A . . . . . . G

G T . . . . . .

T . . . A A A .

T . . . . . . C

A . . G . . . .

0 . . . 1 1 1 1

0 . . . . . 1 .

5. Discussion

H1 H2 H3 H4 H5 H6 H7 H8

329 285 226a 178

trnT-trnL

trnS-trnG

338

424

455

741

819

840

843

850

878

1159

1244

1247b

1248b

1249b

Genetic distance to Polymorphic sites Haplotype

Table 2 Description of the polymorphic sites of the haplotypes found for Pilosocereus and respective genetic distances. (G: guanine, A: adenine, C: cytosine, T: thymine; 1: presence of structural mutation, 0: absence of structural mutation).

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The measure of genetic distance between two lineages or taxa may vary greatly depending on the genetic marker used. Thus, the absence of numerical relationship between genetic distance and spine traits differences is not conclusive. Moreover, if quantitative inheritance genes are indeed involved with spine development and growth, these genes would be directly subjected to natural selection. Therefore, the difference in spine traits would not necessarily show proportionality to genetic distance. Thus, no matter how small, we believe that there must be a genetic component in spine traits, even though, there are obvious inheritable traits such as spine colour and shape, and also because some species have spineless variations (Oliveira et al., 2013). Although, ribs exhibited a moderate relationship with genetic distance, their correlation with latitude was much stronger (see Table 3). Thus, the identification of the genes involved in spine and rib growth as well as their expression is crucial to understand the role of genetics in their morphological traits. Since, spine growth basically depends on cell division and elongation (Mauseth, 1977), and these phenomena are not known to respond directly to solar radiation, no correlation was expected between latitude and spine length. On the other hand, we did expect some relation with rainfall, because minimum water availability is necessary for cell elongation. Under an unlimited water availability spine growth should be limited only by the maturation time of the cell wall and by the activity of the spine’s basal meristematic region. However, under intense drought stress, spine growth would be limited to cell division, since elongation would be minimal. This would explain the positive numeric relationship between spine length and mean annual rainfall. The negative numeric relationship between latitude and number of spines per areole confirms the role of spine shading in reaching optimal radiation levels. However, it was not possible to deduce whether this was to ensure photosynthetic efficiency (Loik, 2008) or to avoid excessive temperatures (Nobel, 1983). In either of these cases, the higher number of spines in low latitudes seems to be a response to excessive radiation, diminishing polewards as radiation decreases. The increase in the number of spines per areole towards the equator can also be a consequence of a higher photosynthetic efficiency allowed by the higher radiation levels. In both cases the number of spines (biomass) is strongly correlated with solar radiation. The negative relationship between the number of spines and mean annual rainfall is concordant with this explanation, since cloud shading diminishes downward radiation, presenting therefore an effect similar to that of high latitudes.

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Table 3 Results of the correlation tests between different variables and traits analyzed (d.f. = degrees of freedom). Variable

Trait

Sampling group

d.f.

p-Value

Spearman’s rank correlation coefficient (rho)

Pair-wise genetic distance

Spine length (pair-wise difference) Number of spines(pair-wise difference) Number of ribs (pair-wise difference) Spine length (mm) Number of spines Number of ribs Spine length (mm) Number of spines Number of ribs

Pilosocereus (7 taxa; 29 samples) Pilosocereus (7 taxa; 29 samples) Pilosocereus (7 taxa; 29 samples) P. arrabidae group(3 taxa; 31 samples) P. arrabidae group(3 taxa; 31 samples) P. arrabidae group(3 taxa; 31 samples) P. arrabidae group(3 taxa; 31 samples) P. arrabidae group(3 taxa; 31 samples) P. arrabidae group(3 taxa; 31 samples)

405 405 405 30 30 30 30 30 30

0.4220 0.9853 <0.0001 0.0007 0.0013 0.8284 0.3811 0.0007 <0.0001

Not significant Not significant +0.4910 +0.5748 −0.5487 Not significant Not significant −0.5722 −0.7457

Mean annual rainfall

Latitude

A negative relationship between the number of ribs and latitude was also expected, since these structures help to promote heat loss (Drezner, 2011) and self-shading of the stem (Mauseth, 2006; Perez-Harguindeguy et al., 2013). This relationship seems to lead to an optimal level of sunlight exposure, as occurs with spine coverage (Loik, 2008). Although, no relationship was found between the number of ribs and rainfall, traits such as rib height and width are very likely to exhibit it, since the the rib dimensions depend on how hydrated the succulent stem is able to become during the rainy season. However, these traits must be sampled in fresh plants, as they are strongly affected by the drying process for making herbarium specimens. Our results and interpretation are in agreement with patterns observed in the albeit scarce literature on this subject. For example, Casas et al. (1999) found longer central spines in Stenocereus stellatus (Pfeiffer) Riccobono grown under in situ management and home cultivation (watered plants) when compared to wild plants (in a semiarid climate). In the same study, cultivated plants presented a higher number of ribs and a higher number of spines per areole when compared with wild plants (a difference that we attribute to the interception of radiation by other plants in the wild environment). Arellano and Casas (2003) mentioned one sample of managed plants of Escontria chiotilla (F.A.C. Weber) Rose with longer spines than those of wild plants (despite the artificial selection of plants with short spines). This is another case that can be attributed to watering under home cultivation. The only apparently discordant data are those from Peharec et al. (2010), where the “hyperhydric” stems of Mammillaria gracilis Pfeiff. presented shorter spines when compared to pot grown plants. However, we stress that their pot-grown plants were in a glasshouse under natural photoperiod and the hyperhydric stems were grown in vitro, under conditions of artificial radiation. Thus, rather than an effect of water availability, we attribute this result to different (ultra-violet) radiation levels. Furthermore, the in-vitro grown stems presented a lower number of spines per areole when compared with the greenhouse plants, corroborating the radiation effect on spine biomass claimed by Loik (2008). Although, we found significant correlations between morphological traits and environmental variables, it is important to consider that we studied wild plants; and that in the natural environment many factors may act synergistically to determine ribs and spine patterns. Although, it is desirable to explore the thresholds of different aspects of radiation and water availability on spine growth, experimental studies with isolation of environmental variables and acceptable quantities of replicates are still scarce. Although not evaluated, the substrate composition is very likely to have some influence on spine growth; if not directly (by the presence or absence of macro- and micronutrients), indirectly by the influence of texture on water availability. Although, it would probably be a secondary role, the type of substrate would still be of some importance, especially when considering horticultural purposes.

6. Conclusions Confirmation of the influence of environmental variables (water availability and solar radiation) on rib and spine patterns of Pilosocereus is by far the most important conclusion of this study. Although challenging, in so far that more studies on this theme are required, perhaps taxonomists should start considering to which extent the use of morphometric traits is reliable for the delimitation and identification of cactus taxa. This consideration is especially important for the use of cultivated specimens as sources of evidence in taxonomy (e.g. Schmalzel et al., 2004; Eggli and Leuenberger, 2008), since water availability and solar radiation in glasshouses may differ remarkably from natural habitats. Acknowledgments Funding for this research was provided by the Fundac¸ão Grupo Boticário de Protec¸ão à Natureza (0970 20131), Conselho Nacional de Desenvolvimento Científico e Tecnológico (522.213/2011–0) and Instituto Chico Mendes de Conservac¸ão da Biodiversidade (Plano de Ac¸ão Nacional para a Conservac¸ão das Cactáceas). The authors would like to thank Felipe Ribeiro, Marlon C. Machado, George M. Tabatinga Filho, Pricila C.M. Aragão, Francisca S. Araújo, Itayguara R. Costa, Diva Correia and Paulo Coelho for the support during field expeditions; Sarah S.G. Souza, Regina C.A. Freitas and Adalberto M.M. Carvalho (EAC) for herbarium specimen preparation. References Arellano, E., Casas, A., 2003. Morphological variation and domestication of Escontria chiotilla (Cactaceae) under silvicultural management in the Tehuacan Valley, Central Mexico. Genet. Resour. Crop Evol. 50, 439–453. Bandelt, H.J., Forster, P., Röhl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Boke, N.H., 1944. Histogenesis of the leaf and areole in Opuntia cylindrica. Am. J. Bot. 31, 299–316. Boke, N.H., 1980. Developmental morphology and anatomy in Cactaceae. BioScience 30, 605–610. Bonatelli, I.A.S., Zappi, D.C., Taylor, N.P., Moraes, E.M., 2013. Applicability of plastid DNA regions for intra- and interspecific studies in closely related cacti species. Genet. Mol. Res. 12, 4579–4585. Bonatelli, I.A.S., Perez, M.F., Peterson, T., Taylor, N.P., Zappi, D.C., Machado, M.C., Koch, I., Pires, A., Moraes, E.M., 2014. Interglacial microrefugia and diversification of a cactus species complex: phylogeography and palaeodistributional reconstructions for Pilosocereus aurisetus and allies. Mol. Ecol. 23, 3044–3063. Casas, A., Caballero, J., Valiente-Banuet, A., Soriano, J.A., Dávila, P., 1999. Morphological variation and the process of domestication of Stenocereus stellatus (Cactaceae) in Central Mexico. Am. J. Bot. 86, 522–533. Drezner, T.D., 2011. Cactus surface temperatures are impacted by seasonality, spines, and height on plant. Environ. Exp. Bot. 74, 17–21. Edgar, R.C., 2004. Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Eggli, U., Leuenberger, B.E., 2008. Type specimens of Cactaceae names in the Berlin Herbarium (B). Willdenowia 38, 213–280. Guerrero, P.C., Carvallo, G.O., Nassar, J.M., Rojas-Sandoval, J., Sanz, V., Medel, R., 2012. Ecology and evolution of negative and positive interactions in Cactaceae: lessons and pending tasks. Plant Ecolog. Divers. 5, 205–215.

M.O.T. Menezes et al. / Flora 214 (2015) 44–49 Hatzianastassiou, N., Matsoukas, C., Fotiadi, A., Pavlakis, K.G., Drakakis, E., Hatzidimitriou, D., Vardavas, I., 2005. Global distribution of Earth’s surface shortwave radiation budget. Atmos. Chem. Phys. 5, 2847–2867. Hunt, D., Taylor, N., Charles, G., 2006. The New Cactus Lexicon. DH Books, Milborne Port. Loik, M.E., 2008. The effect of cactus spines on light interception and photosystem II for three sympatric species of Opuntia from the Mojave Desert. Physiol. Plant. 134, 87–98. L.C. Majure, 2007, The ecology and morphological variation of Opuntia (Cactaceae) species in the mid-south, United States, Unpublished Master Thesis (Mississipi State University), Available at: Unpublished http://www.gri.msstate.edu/ publications/docs/2007/08/3887Majure Thesis.pdf Mauseth, J.D., 1977. Cytokinin- and gibberellic acid-induced effects on the determination and morphogenesis of leaf primordia in Opuntia polyacantha (Cactaceae). Am. J. Bot. 64, 337–346. Mauseth, J.D., 2006. Structure-function relationships in highly modified shoots of Cactaceae. Ann. Bot. 98, 901–926. Mihalte, L., Sestras, R.E., 2012. The plant size and the spine characteristics of the first generation progeny obtained through the cross–pollination of different genotypes of Cactaceae. Euphytica 184, 369–376. Nobel, P.S., 1983. Spine influences on par interception, stem temperature, and nocturnal acid accumulation by cacti. Plant Cell Environ. 6, 153–159.

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Oliveira, F.I.C., Bordallo, P.N., Castro, A.C.R., Correia, D., 2013. Genetic diversity of spineless Cereus jamacaru accessions using morphological and molecular markers. Genet. Mol. Res. 12, 4586–4594. Peharec, P., Posilovic, H., Balen, B., Krsnik-Rasol, M., 2010. Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots. J. Microsc. 239, 78–86. Perez-Harguindeguy, N., Diaz, S., Garnier, E., Lavorel, S., Poorter, H., Jaureguiberry, P., Bret-, M.S., Harte, Cornwell, W.K., Craine, J.M., Gurvich, D.E., Urcelay, C., Veneklaas, E.J., Reich, P.B., Poorter, L., Wright, I.J., Ray, P., Enrico, L., Pausas, J.G., de Vos, A.C., Buchmann, N., Funes, G., Quetier, F., Hodgson, J.G., Thompson, K., Morgan, H.D., ter Steege, H., van der Heijden, M.G.A., Sack, L., Blonder, B., Poschlod, P., Vaieretti, M.V., Conti, G., Staver, A.C., Aquino, S., Cornelissen, J.H.C., 2013. New handbook for standardised measurement of plant functional traits worldwide. Aust. J. Bot. 61, 167–234. Core Team, R., 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project. org Rowley, G.D., 2003. What is an areole? Brit. Cact. Succ. J. 21, 4–11. Sajeva, M., Mauseth, J.D., 1991. Leaf-like structure in the photosynthetic, succulent stems of cacti. Ann. Bot. 68, 405–411. Schmalzel, R.J., Nixon, R.T., Best, A.L., Tress Jr., J.A., 2004. Morphometric variation in Coryphantha robustispina (Cactaceae). Syst. Bot. 29, 553–568. Zappi, D., 1994. Pilosocereus (Cactaceae): The Genus in Brazil. Royal Botanical Gardens, Kew.