The Bromeliaceae are exclusive to the tropical and subtropical areas of the Americas (except for one African species) and show extreme adaptive radiation. Species of this typical neotropical family have evolved to fill a variety of niches in a highly heterogeneous habitat, with an amazing diversity of adaptations ([@bib2]). Such rapid processes of adaptation and speciation can be used as models to study and understand larger issues regarding the evolution of neotropical plants. Studies on bromeliad species adapted to neotropical rock outcrops have improved our understanding of the processes of speciation and species cohesion in naturally isolated environments (i.e., [@bib1]; [@bib12]).

*Orthophytum* Beer species are rupicolous herbs exclusive to the northeastern and southeastern regions of Brazil ([@bib10]). Although 2*n* = 50 chromosomes are common within the Bromeliaceae, many *Orthophytum* species are polyploids (2*n* = 100 and 150) ([@bib4]; [@bib8]). Species of this genus generally inhabit granitic-gneiss inselbergs and quartzitic-sandstone outcrops in the Brazilian campos rupestres ("rocky fields") along the Espinhaço Range ([@bib9]). Such environments are spatially and ecologically isolated, providing a barrier against dispersal and migration, and they possess elevated species diversity and high levels of endemism ([@bib6]). In fact, most *Orthophytum* species are restricted to small geographic ranges and some of them are narrow endemics ([@bib10]). This genus offers an interesting model system for examining speciation processes and endemism in neotropical mountainous habitats.

Here, we report microsatellite loci isolation, characterization, and cross-amplification for *O. ophiuroides* Louzada & Wand., a species endemic to the Chapada Diamantina section of the Espinhaço Range in the state of Bahia, Brazil ([@bib9]). The microsatellite loci reported here will be used to access levels of gene flow to resolve forces operating during species radiations on naturally isolated habitats. The knowledge of patterns of diversity and gene flow will also be important for management of conservation strategies and for understanding the genetic consequences of variation in mating systems or pollination syndromes.

METHODS AND RESULTS
===================

To characterize microsatellite loci, 42 individuals of *O. ophiuroides* located in two populations from Lençóis in the Brazilian Federal State of Bahia were collected and stored in silica gel. Genomic DNA was extracted from leaf tissue using the protocol described by [@bib11].

The microsatellite-enriched library of *O. ophiuroides* was constructed according to [@bib3]. Genomic DNA was digested using the restriction enzyme *Rsa*I (Invitrogen, Carlsbad, California, USA). The library was then enriched for (CT)~8~ and (GT)~8~ repeats using biotinylated microsatellite probes, and the target fragments were captured by the use of streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA). These microsatellite-enriched DNA fragments were cloned into pGEM-T Easy Vector (Promega Corporation), and competent XL1-blue *Escherichia coli* cells were transformed with the recombinant plasmids. The resulting recombinant clones were sequenced using the BigDye Terminator Cycle Sequencing Kit (version 3.1) on an ABI 3730 DNA Analyzer Sequencer (Applied Biosystems, Carlsbad, California, USA).

For the clones containing microsatellite motifs, forward and reverse sequences were aligned using the software Sequencher version 4.1.2 (Gene Codes Corporation, Ann Arbor, Michigan, USA), and primers were designed with Primer3 software ([@bib14]). Forward primers were synthesized with a 19-bp M13 tail (5′-CACGACGTTGTAAAACGAC-3′) at the 5′ end to allow labeling with a tailed fluorescent dye M13 primer during amplification and genotyping procedures.

The microsatellite fragments were PCR amplified in a reaction volume of 10 μL containing ∼20 ng DNA template, 5× GoTaq Master Mix (Promega Corporation), 5 pmol forward primer, 10 pmol reverse primer, and 1 pmol universal M13 primer tagged with distinct fluorochromes (FAM, VIC, PET, or NED). PCRs were performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems) using different touchdown or standard cycling programs according to the annealing temperature of the primers ([Table 1](#tbl1){ref-type="table"}) as described by [@bib11]. Loci were genotyped on an ABI 3730 DNA Analyzer Sequencer and sized against a GeneScan LIZ molecular size standard (Applied Biosystems) using GeneMarker software v.DEMO (SoftGenetics, State College, Pennsylvania, USA).

###### 

Primer sequences and characteristics of microsatellite loci from *Orthophytum ophiuroides*.

  Locus   Primer sequences (5′--3′)    Repeat motif                      *T*~a~ (°C)[^a^](#tblfn1){ref-type="table-fn"}   Allele size range (bp)   GenBank accession no.
  ------- ---------------------------- --------------------------------- ------------------------------------------------ ------------------------ -----------------------
  Op13    F: CACCCATTGAGAAGGAAACG      (CA)~12~                          TD-48                                            208--216                 KF753870
          R: GATGAGTGGACTGGTAAAGACC                                                                                                                
  Op30    F: CAACCCTTTTTGTGCCCACT      (CT)~24~                          ST-54                                            140--185                 KF753871
          R: CACTTGTGATGTTTCCTCGTC                                                                                                                 
  Op38    F: TGCCCACTGTGCTGCTATTA      (TCA)~8~N(GT)~9~(GA)~10~          ST-54                                            162--182                 KF753872
          R: GAGCTCATCCATGGCGATT                                                                                                                   
  Op62    F: ATAAGGCCCTCTCGAATGCT      (CT)~27~AT(CTAT)~6~CT(GT)~9~      TD-48                                            190--233                 KF753873
          R: GCATGTAAACCAGCCAACCT                                                                                                                  
  Op69    F: CCTCCCTTTTTCCCTCCTAC      (CT)~20~                          TD-48                                            108--136                 KF753874
          R: ACGCGCTCTACTACCACCAT                                                                                                                  
  Op73    F: ACCATCGTCCTGTTCACGTC      (AC)~16~                          ST-54                                            168--189                 KF753875
          R: CAGCTAAACTAAGAGAGGGTGGA                                                                                                               
  Op77    F: CACAACAGAGGCTCGAAAGA      (AG)~10~                          TD-48                                            151--168                 KF753876
          R: GCCCGACTCCTCCAATAACC                                                                                                                  
  Op78    F: CCGGAAAAGCTGTAATGTTTC     (CT)~9~A(CT)~8~(CT)~12~(CA)~17~   TD-48                                            263--299                 KF753877
          R: GCGTGGCTCTCTTTTATGGT                                                                                                                  
  Op89    F: AACCCTAGTTCCACCGATCA      (CT)~11~                          TD-48                                            135--139                 KF753878
          R: CTCTTCACCCCCACAAATTC                                                                                                                  
  Op92    F: TCTTTCTCCGTTCAGCTCTCA     (CT)~7~CC(CT)~9~CC(CT)~6~         TD-48                                            271--287                 KF753879
          R: CCGGTTTCCAGGATCAATAA                                                                                                                  
  Op93    F: TTATCGGGCAGGGGAAATTA      (CT)~12~(GT)~8~                   TD-48                                            161--178                 KF753880
          R: ACCTTGTCACACACGCAAAG                                                                                                                  

*Note*: *T*~a~ = annealing temperature.

TD = touchdown program cycling as published by [@bib11]; ST = standard program cycling: 95°C for 3 min, 40 cycles of 94°C for 30 s, 54°C for 30 s, 72°C for 30 s, and a final elongation step at 72°C for 10 min.

The levels of genetic diversity were evaluated through the following descriptive summary statistics: expected heterozygosity (*H*~e~), observed heterozygosity (*H*~o~), and number of alleles per locus (*A*). These parameters were calculated with the MSA program ([@bib5]). Tests for departure from Hardy--Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were calculated for all the polymorphic loci with GENEPOP version 3.5 ([@bib13]). For population 2, which showed a tetraploid genotyping pattern, genetic diversity parameters were calculated with AUTOTET, specific to polyploid species ([@bib15]). The same program was used to test for departure from HWE for the polyploid population.

Cross-amplification tests were performed for nine bromeliad species from three subfamilies ([Appendix 1](#tbla1){ref-type="table"}) using the same protocols established for each loci: *O. disjunctum* L. B. Sm., *O. foliosum* L. B. Sm., and *O. zanonii* Leme (Bromelioideae); *Vriesea gigantea* Mart. ex Schult. f. and *V. scalaris* E. Morren (Tillandsioideae); and *P. albiflos* Herb., *P. corcovadensis* Wawra, *P. flammea* Lindl., and *P. staminea* Lodd. (Pitcarnioideae).

From the 96 sequenced recombinant colonies, 78 contained microsatellite motifs and 29 of those presented proper flanking regions for primer design ([Table 1](#tbl1){ref-type="table"}). Eleven microsatellite loci amplified and were polymorphic for population 1 while nine loci amplified and were polymorphic for population 2 ([Table 2](#tbl2){ref-type="table"}). Loci that performed poorly in the amplification screening or were monomorphic are listed in Appendix S1. In the genotyping process, population 1 showed a diploid and codominant pattern, with no more than two bands per individual, while population 2 showed a tetraploid and codominant pattern, with up to four bands per individual. Overall, the 11 microsatellite loci were highly polymorphic. The number of alleles per locus ranged from five to nine in population 1, and from three to nine in population 2 ([Table 2](#tbl2){ref-type="table"}). *H*~e~ and *H*~o~ ranged from 0.167 to 0.870 and from 0.369 to 0.958, respectively. The inbreeding coefficient values were high for most of the loci, and eight of them showed significant departures from HWE in population 1, while all of the loci showed significant departures in population 2. LD was significant (*P* \< 0.05) for 33 out of 55 pairwise locus comparisons. Such departures (HWE and LD) were consistent with inbreeding and/or Wahlund effects. Although null alleles cannot be ruled out, MICRO-CHECKER software ([@bib17]) found no evidence for scoring error due to stuttering or large allele dropout. Because of the polyploidy of population 2, MICRO-CHECKER could only be used to estimate the null alleles in population 1. The differences in the number of alleles per locus between the two populations indicate that different cytotypes may occur within *O. ophiuroides* species, a phenomenon observed for other plants ([@bib7]) including bromeliad species (i.e., *Tillandsia*: [@bib16]).

###### 

Characteristics of microsatellite loci from two populations of *Orthophytum ophiuroides*.[^a^](#tblfn2){ref-type="table-fn"}

         Population 1 (*n* = 26)   Population 2 (*n* = 18)                                                                  
  ------ ------------------------- ------------------------- ------- ------- ------------- ---------- ----- ------- ------- --------------
  Op13   208--216                  5                         0.190   0.524   0.642\*\*\*   208--216   5     0.686   0.410   0.402\*\*\*
  Op30   140--185                  9                         0.583   0.832   0.304\*\*\*   ---        ---   ---     ---     ---
  Op38   162--182                  5                         0.467   0.772   0.404\*\*     ---        ---   ---     ---     ---
  Op62   190--233                  8                         0.870   0.854   −0.019        190--218   7     0.831   0.958   −0.153\*
  Op69   108--136                  9                         0.714   0.791   0.099         108--123   4     0.723   0.708   0.021\*\*
  Op73   168--189                  6                         0.400   0.677   0.415\*\*     152--191   6     0.749   0.369   0.507\*\*\*
  Op77   151--168                  6                         0.571   0.762   0.255\*\*     151--162   5     0.752   0.778   −0.034\*\*
  Op78   263--299                  8                         0.826   0.830   0.005\*\*     265--303   9     0.848   0.912   −0.076\*
  Op89   135--139                  5                         0.167   0.708   0.770\*\*\*   125--139   3     0.567   0.469   0.173\*\*\*
  Op92   271--287                  7                         0.750   0.849   0.119         268--293   7     0.806   0.885   −0.097\*\*\*
  Op93   161--178                  5                         0.286   0.608   0.540\*\*     139--194   6     0.682   0.556   0.185\*\*\*

*Note*: *A* = number of alleles; *f* = inbreeding coefficient; *H*~e~ = expected heterozygosity; *H*~o~ = observed heterozygosity.

Geographic coordinates of the populations: Population 1 = 12°34′S, 41°22′W and Population 2 = 12°30′S, 40°25′W. Voucher information: Population 1 = Louzada & Wanderley 88 (SP) and Population 2 = Louzada 194 (UFP).

Significant departure from Hardy--Weinberg equilibrium: \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001.

Seven of the markers showed satisfactory amplification within the expected allele size range across three Bromeliaceae subfamilies in the cross-amplification tests (Op13, Op30, Op62, Op69, Op77, Op89, and Op93) ([Table 3](#tbl3){ref-type="table"}). This indicates that this set of loci could be used in population genetic studies involving species cohesion and delimitation, phylogeography, and barriers to gene flow for other bromeliad species.

###### 

Cross-amplification of seven microsatellite loci isolated from *Orthophytum ophiuroides*.

                                 Allele size range (bp)                                                               
  ------------------------------ ------------------------ ---------- ---------- ---------- --------------- ---------- -----
  *Vriesea gigantea* (2)         212--217                 ---        ---        ---        151--153--161   136--140   168
  *V. scalaris* (2)              212                      ---        ---        114        151--161        138--140   ---
  *Pitcairnia albiflos* (2)      212                      140--150   ---        ---        151--153--161   136--140   168
  *P. corcovadensis* (2)         212                      ---        196--206   ---        151--153        136--140   168
  *P. flammea* (2)               212                      ---        206        112--114   151--153        136--140   168
  *P. staminea* (2)              212                      138--152   206        114--136   151--159--161   136        168
  *Orthophytum disjunctum* (1)   212                      138--139   175--206   ---        183             125--136   168
  *O. foliosum* (1)              233                      142--165   189--219   ---        188--190        136        ---
  *O. zanonii* (1)               233                      144--148   ---        151        203             140--147   168

CONCLUSIONS
===========

The data showed that the set of optimized and tested microsatellite markers will be useful for studying evolution and population genetics of *O. ophiuroides* and other species from the same genus. Furthermore, the markers can also be used for studying species within other genera of the Bromeliaceae family.

Supplementary Material
======================

###### 

Click here for additional data file.

###### 

Sampling information, locality details, and voucher information for analyzed samples.

  Species                                  Locality                            Voucher (Herbarium)[^a^](#tblfn4){ref-type="table-fn"}
  ---------------------------------------- ----------------------------------- --------------------------------------------------------
  *Vriesea gigantea* Mart. ex Schult. f.   Itaimbezinho, RS, Brazil            Martinelli et al. 14897 (RB)
  *V. scalaris* E. Morren                  Santa Lucia, ES, Brazil             Coser 4070 (MBML)
  *Pitcairnia albiflos* Herb.              Rio de Janeiro, RJ, Brazil          Wendt 593 (RB)
  *P. corcovadensis* Wawra                 Rio de Janeiro, RJ, Brazil          Wendt 18 (RB)
  *P. flammea* Lindl.                      Campina Grande do Sul, PR, Brazil   Gasper 300 (FURB)
  *P. staminea* Lodd.                      Rio de Janeiro, RJ, Brazil          Wendt 143 (RB)
  *Orthophytum disjunctum* L. B. Sm.       Pesqueira, PE, Brazil               Louzada et al. 149 (SP)
  *O. foliosum* L. B. Sm.                  Santa Teresa, ES, Brazil            Louzada et al. 13 (SP)
  *O. ophiuroides* Louzada & Wand.         Lençóis, BA, Brazil                 Louzada & Wanderley 88 (SP)
  *O. zanonii* Leme                        Pancas, ES, Brazil                  Louzada et al. 18 (SP)

*Note*: BA = Bahia; ES = Espírito Santo; PE = Pernambuco; PR = Paraná; RJ = Rio de Janeiro; RS = Rio Grande do Sul.

Herbarium codes: FURB = Herbário Dr. Roberto Miguel Klein; MBML = Herbário Mello Leitão; RB = Herbário Dimitri Sucre Benjamin; SP = Herbário do Estado "Maria Eneyda P. Kaufmann Fidalgo".

[^1]: The authors thank Anete Pereira de Souza for assistance during library construction. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2011/06438-2 to F.A-G., 2009/52725-3 and 2009/17411-8 to C.P-S.).
