**5. Molecular studies in** *Alstroemeria*

the different taxa, as well as to the understanding of the chromosomal processes that determine the divergence among them [39]. Recent studies at the infraspecific level, in taxonomic complexes of the genus, have also been shown to be useful in the recognition of these taxa, either due to differences in the chromosomal architecture or in the asymmetry indexes of the chromosomes [40, 41]. Strasburger [42] was the first researcher to perform chromosome studies in *Alstroemeria* and until 1989 the number of cytological published papers involved no more than 10 different species [43]. In the last 25 years, a wide variety of cytogenetic studies have been carried out in the genus, including physical location of repetitive DNA sequences in *A. aurea* [16, 44, 45], meiosis and mitosis [46], karyology [47–49], variation and size of the genome [50], fluorescent *in situ* hybridization [39, 51, 52] and cytotaxonomy [40, 41, 52–58]. In 15 geographically isolated populations of five species of *Alstroemeria* (*A. aurea*, *A. hookeri*, *A. ligtu*, *A. pelegrina* and *A. presliana*) collected in Chile, karyotypes and variation of RAPD markers have been investigated. Tandemly repeated DNA sequences—5S and 18/25S rDNA genes and the sequence A001-1 were used to characterize karyotypes by fluorescence in situ hybridization (FISH). Ten somatic metaphases per population were used for measurement of chromosome length. Differences in RAPD marker bands were used for characterization of populations, creating a similarity index. FISH with all three DNA probes shows a high degree of polymorphism among and sometimes also within accessions of *A. aurea*, *A. hookeri* and *A. ligtu*. The number of chromosome pairs showing 5S rDNA signals is more different for the investigated species *A. aurea*, *A. hookeri*, *A. ligtu*, *A. pelegrina* and *A. presliana* with 5, 7, 5, 3 and 7, respectively, than the number of 18/25S rDNA signals in this succession with 7, 7, 6, 5 and 7 chromosome pairs, showing a high evolutionary dynamics within the genus. Furthermore, among the four populations of *A. hookeri*, accession 4181 was different in arm length of chromosome 3. RAPD markers (index of similarity) also showed a greater genetic

242 Selected Studies in Biodiversity

distance of accession 4181 from the other three accessions of *A. hookeri* [39].

The study of the chromosomes in *Alstroemeria* has already helped to clarify a number of taxonomic issues within the genus. For example, study of karyotypes in the *A. hookeri* complex permitted change a subspecies to the species rank (*A. cummingiana*), the recognition of a new subspecies (*A. hookeri* subsp. *sansebastiana*) and description of a new species (*A. marticorenae*) [19, 22, 40, 41]. Similar situation occurred in the *A. presliana* complex, where after completing a comparative karyotypic study in 11 populations, it was suggested that *A. presliana* subsp. *australis*, endemic to the cordillera of Nahuelbuta, should be raised to species rank [18].

A number of cytological studies have been completed in the *Alstroemeria ligtu* complex. Buitendijik and Ramanna [16] and Buitendjik et al. [50] found variation in the DNA content and polymorphism of C bands in the chromosomes of subsp. *ligtu*, subsp. *simsii* and subsp. *splendens*. Zhou et al. [51], utilizing FISH, completed the characterization of the genomic DNA of eight highly repetitive sequences in subsp. *ligtu* and *simsii*, showing detailed karyotypes with localization of specific DNA sequences. DAPI staining and acetic orcein, completed a comparative karyotype study of five populations of subsp. *ligtu* from the Region of Biobío and one population of subsp. *simsii* from the Region of Valparaiso [39]. The six populations studied revealed an asymmetric karyotype with 2n = 2x = 16 chromosomes. The populations of subsp. *ligtu* have a haploid formula of four metacentric chromosomes (chromosomes 1 and 2 with microsatellites), one submetacentric with a microsatellite and three telocentric

During recent years, an increasing accessibility to molecular data and the development of a vast range of bioinformatics analysis has favored the successful implementation of genetic tools in the identification and conservation of biological diversity [59]. Dominant molecular markers based on random fragment alleles e.g. Inter Simple Sequence Repeat (ISSR), Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) have been used for characterizing genetic diversity in *Alstroemeria* [60–62], becoming the marker of choice for the identification of cultivar varieties with ornamental value [63–65] and conducting population genetic analyses [66, 67]. The use of DNA sequences has been more related to the construction of phylogenetic hypotheses and establishment of biogeographic patterns [8, 9]. Interestingly, near 30% of the Chilean species of *Alstroemeria* form species complexes, comprising from two to four infraspecific taxa each. This pattern is likely explained by adaptation to a wide range of environmental heterogeneity present in Chile [68], which is possibly driving processes of microevolutionary divergence [14]. Given the complexity of interpreting the integrity within and among these species complexes, we started several initiatives for applied genetic studies with the purpose of disentangling the discernibility of intraspecific patterns of divergence, especially in groups highly regarded for their ornamental and conservation value.

#### **5.1. Molecular markers in assessing genetic diversity for conservation in** *Alstroemeria*

A priority goal in conservation is to evaluate levels of apportionment of genetic diversity in targeted species, given the association between population genetic diversity and their potential for local adaptation and evolutionary resilience. Genetic variability is the result of the dynamics of gene flow, for which a homogeneous distribution of allelic frequencies is expected under high levels of gene flow among populations [69]. Interestingly, this situation is rarely found in nature, since the strong effect that geographic isolation and selection represents for local populations of plants. As a result, it is not surprising that peripheral populations tend to increase gene differentiation and population structure levels; hence, contributing to the local isolation that eventually could result in different isolated species (**Figure 7**) [69].

Such patterns of isolation and divergence are no exception in *Alstroemeria*, for which high levels of structuration are documented. For example, *Alstroemeria hookeri* represents a species complex that comprises four subspecies, two of them (subspecies *recumbens* and *maculata*) distributed in North-Central of Chile and two (subspecies *hookeri* and *sansebastiana*) in southern Chile. Based on ISSR (Inter Simple Sequence Repeat) markers, high levels of population structure were found among southern subspecies (**Figure 8**, **Table 2**); also concomitant with previous findings found with allozymes markers [67]. Similarly, high levels of within population diversity was found in *A. presliana* complex using AFLP markers (**Table 2**), exhibiting significant levels of among population variability and moderate levels of genetic population structure (**Table 2**). This complex comprises of two varieties (var. *presliana* and var. *australis*), both separately distributed across Coastal and Andean mountain ranges in Chile (**Figure 9**). The results from both complexes showed the existence of two heterogeneous genetic groups with no evident spatial congruence suggesting genetic differentiation among varieties or subspecies. Interestingly, several populations are individually differentiated in their genetic profiles, despite of occurring closely enough with other neighbored populations to sustain substantial levels of gene flow (**Figure 9**).

Among explanations of the observed patterns of genetic diversity found in *Alstroemeria*, strategies of reproduction and dispersal become plausible enough to be considered. *Alstroemeria* species have a restricted capacity of seed and pollen dispersal [66], which in combination with their vegetative reproduction by rhizomes [14, 67], could contribute to maintaining restricted levels of gene flow and sustaining high levels of genetic structure

**Figure 7.** Cytogenetic studies in Chilean *Alstroemeria*. (A) Mitotic metaphase of *Alstroemeria hookeri* Ssp. *hookeri.* (B) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using 5S genes. (C) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using 18-25S genes. (D) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using A001 genes. (E) Ideogramm of *Alstroemeria hookeri* ssp. *hookeri* showing genes 5S, 18-25S, and A001 (FISH). (F) Karyotypes of *Alstroemeria presliana*:

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above, *A. presliana* ssp. *presliana*; below, *A. presliana* ssp. *australis.*

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been used for characterizing genetic diversity in *Alstroemeria* [60–62], becoming the marker of choice for the identification of cultivar varieties with ornamental value [63–65] and conducting population genetic analyses [66, 67]. The use of DNA sequences has been more related to the construction of phylogenetic hypotheses and establishment of biogeographic patterns [8, 9]. Interestingly, near 30% of the Chilean species of *Alstroemeria* form species complexes, comprising from two to four infraspecific taxa each. This pattern is likely explained by adaptation to a wide range of environmental heterogeneity present in Chile [68], which is possibly driving processes of microevolutionary divergence [14]. Given the complexity of interpreting the integrity within and among these species complexes, we started several initiatives for applied genetic studies with the purpose of disentangling the discernibility of intraspecific patterns of divergence, especially in groups highly regarded for their ornamental and conservation value.

A priority goal in conservation is to evaluate levels of apportionment of genetic diversity in targeted species, given the association between population genetic diversity and their potential for local adaptation and evolutionary resilience. Genetic variability is the result of the dynamics of gene flow, for which a homogeneous distribution of allelic frequencies is expected under high levels of gene flow among populations [69]. Interestingly, this situation is rarely found in nature, since the strong effect that geographic isolation and selection represents for local populations of plants. As a result, it is not surprising that peripheral populations tend to increase gene differentiation and population structure levels; hence, contributing to the local isolation that eventually could result in different isolated species (**Figure 7**) [69]. Such patterns of isolation and divergence are no exception in *Alstroemeria*, for which high levels of structuration are documented. For example, *Alstroemeria hookeri* represents a species complex that comprises four subspecies, two of them (subspecies *recumbens* and *maculata*) distributed in North-Central of Chile and two (subspecies *hookeri* and *sansebastiana*) in southern Chile. Based on ISSR (Inter Simple Sequence Repeat) markers, high levels of population structure were found among southern subspecies (**Figure 8**, **Table 2**); also concomitant with previous findings found with allozymes markers [67]. Similarly, high levels of within population diversity was found in *A. presliana* complex using AFLP markers (**Table 2**), exhibiting significant levels of among population variability and moderate levels of genetic population structure (**Table 2**). This complex comprises of two varieties (var. *presliana* and var. *australis*), both separately distributed across Coastal and Andean mountain ranges in Chile (**Figure 9**). The results from both complexes showed the existence of two heterogeneous genetic groups with no evident spatial congruence suggesting genetic differentiation among varieties or subspecies. Interestingly, several populations are individually differentiated in their genetic profiles, despite of occurring closely enough

with other neighbored populations to sustain substantial levels of gene flow (**Figure 9**).

Among explanations of the observed patterns of genetic diversity found in *Alstroemeria*, strategies of reproduction and dispersal become plausible enough to be considered. *Alstroemeria* species have a restricted capacity of seed and pollen dispersal [66], which in combination with their vegetative reproduction by rhizomes [14, 67], could contribute to maintaining restricted levels of gene flow and sustaining high levels of genetic structure

**5.1. Molecular markers in assessing genetic diversity for conservation** 

**in** *Alstroemeria*

244 Selected Studies in Biodiversity

**Figure 7.** Cytogenetic studies in Chilean *Alstroemeria*. (A) Mitotic metaphase of *Alstroemeria hookeri* Ssp. *hookeri.* (B) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using 5S genes. (C) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using 18-25S genes. (D) Mitotic metaphase of *Alstroemeria hookeri* ssp. *hookeri* using A001 genes. (E) Ideogramm of *Alstroemeria hookeri* ssp. *hookeri* showing genes 5S, 18-25S, and A001 (FISH). (F) Karyotypes of *Alstroemeria presliana*: above, *A. presliana* ssp. *presliana*; below, *A. presliana* ssp. *australis.*

**Figure 8.** Population structure inferred with ISSR in the *A. hookeri* complex. Bar plots colors represent levels of genetic membership (k = 2) in each individual per sampled population, as inferred under Bayesian admixture inference criterion with the program STRUCTURE [70].

among populations [67]. The sum of these factors implies that local populations could be subject to strong geographic and ecological isolation, which would explain the diversity of infraspecific taxa found in this and other species complexes [67]. In general, moderate to high levels of among population genetic diversity were detected in the studied *Alstroemeria* species complexes. From a conservation perspective, this pattern suggests that protection

initiatives should consider as many populations as possible, in order to preserve the largest

**Figure 9.** Population structure inferred with AFLP in the *A. presliana* Complex. Bar plots colors represent levels of genetic membership (k = 2) in each individual per sampled population, as inferred under Bayesian admixture inference criterion

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Phylogenetic studies provide a theoretical framework to understand the relationships among populations and species. It is desirable that taxa must represent monophyletic lineages, thus reflecting the genetic, evolutionary and biogeographical integrity of lineages and species. Under such premise, several phylogenetic studies in *Alstroemeria* have been conducted integrating a diverse array of molecular, morphological and cytological data [8, 9]. Chacón et al. [8] conducted the most recent and comprehensive phylogenetic studies in genus *Alstroemeria*, based on DNA sequences and cytological data. From taxonomic and evolutionary perspective, three are the most relevant results: (1) Samples belonging to the same species were retrieved as monophyletic; (2) a biogeographic break exists between Brazilian and Chilean species groups and (3) a relatively recent divergence has occurred with the most species of the genus, being diverged during the last 8 millions of years. Interestingly, some of these results have been confirmed from previous initiatives, especially those reflecting the monophyly of

the Brazilian species group with alternative molecular markers (i.e., AFLP) [61].

proportion of total species genetic diversity.

with the program STRUCTURE [70].

**5.2. Molecular phylogenetic studies in** *Alstroemeria*


**Table 2.** Genetic diversity values obtained with allozymes and DNA markers (fragments analyses), for *A. hookeri* and *A. presliana* complexes.

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**Figure 9.** Population structure inferred with AFLP in the *A. presliana* Complex. Bar plots colors represent levels of genetic membership (k = 2) in each individual per sampled population, as inferred under Bayesian admixture inference criterion with the program STRUCTURE [70].

initiatives should consider as many populations as possible, in order to preserve the largest proportion of total species genetic diversity.

#### **5.2. Molecular phylogenetic studies in** *Alstroemeria*

among populations [67]. The sum of these factors implies that local populations could be subject to strong geographic and ecological isolation, which would explain the diversity of infraspecific taxa found in this and other species complexes [67]. In general, moderate to high levels of among population genetic diversity were detected in the studied *Alstroemeria* species complexes. From a conservation perspective, this pattern suggests that protection

**Within pop. Among pop.**

**Species Subspecies He Fst AMOVA (%) Marker/source**

*A. hookeri hookeri* 0.052 0.582 41.71 58.28 Allozymes/Ruiz et al. [67]

*A. presliana presliana* 0.200 0.171 82.91 17.0 AFLP/unpublished

*hookeri* 0.248 0.415 58.47 41.53 ISSR/unpublished *sansebastiana* 0.246 0.36 63.99 36.01 ISSR/unpublished

*australis* 0.198 0.179 82.10 17.9 AFLP/unpublished

**Table 2.** Genetic diversity values obtained with allozymes and DNA markers (fragments analyses), for *A. hookeri* and

**Figure 8.** Population structure inferred with ISSR in the *A. hookeri* complex. Bar plots colors represent levels of genetic membership (k = 2) in each individual per sampled population, as inferred under Bayesian admixture inference criterion

with the program STRUCTURE [70].

246 Selected Studies in Biodiversity

*A. presliana* complexes.

Phylogenetic studies provide a theoretical framework to understand the relationships among populations and species. It is desirable that taxa must represent monophyletic lineages, thus reflecting the genetic, evolutionary and biogeographical integrity of lineages and species. Under such premise, several phylogenetic studies in *Alstroemeria* have been conducted integrating a diverse array of molecular, morphological and cytological data [8, 9]. Chacón et al. [8] conducted the most recent and comprehensive phylogenetic studies in genus *Alstroemeria*, based on DNA sequences and cytological data. From taxonomic and evolutionary perspective, three are the most relevant results: (1) Samples belonging to the same species were retrieved as monophyletic; (2) a biogeographic break exists between Brazilian and Chilean species groups and (3) a relatively recent divergence has occurred with the most species of the genus, being diverged during the last 8 millions of years. Interestingly, some of these results have been confirmed from previous initiatives, especially those reflecting the monophyly of the Brazilian species group with alternative molecular markers (i.e., AFLP) [61].

Despite the promising of these results, species from the Chilean group were mostly underrepresented, making difficult to obtain relevant evidence of local patterns of diversification, particularly to those depicting evolutionary trends or taxonomic integrity in species complexes. Nonetheless, while some progress has been achieved scrutinizing chloroplast sequences (*rpl*32-*trn*l), discordant results challenge the hypothesis integrity previously stated in several of these groups. While *Alstroemeria hookeri* and *A. presliana* complexes are retrieved as monophyletic clades, other groups like *A. magnifica* and *A. ligtu* are retrieved as paraphyletic (**Figure 10**).

divergent patterns of genetic variation are the direct result of breaks in gene flow, leading to phenotypic and genotypic differences that sustain isolated and differentiated species and populations [69]. The simultaneous use of multiple molecular markers and criteria of delimitation has improved the taxonomic work, particularly helping to contextualize the role of microevolutionary processes in the species generation. As previously stated, species of *Alstroemeria* share attributes that could heavily influence patterns of micro evolutionary isolation and divergence, such as restricted seed and pollen dispersal and vegetative reproduction by rhizomes. Therefore, the wide distribution of taxonomic complexes in areas with contrasting topography and climatic conditions implies the existence of restrictions for gene flow, where substantial effects of ecogeographic isolation and divergence are expected in local diversification patterns [67]. Hence, micro evolutionary processes are currently underway and active [14], probably producing decoupled or unnoticeable patterns of divergence. Traditional taxonomic treatment in *Alstroemeria* has heavily relied on the interpretation of floral diversity and vegetative attributes, which has resulted in an important number of recognized taxa and species complexes described [14]. Nonetheless, because active of micro-evolutionary divergence may not ensure congruence among the diverse phenotypic and genotypic characters, it is likely that an under or overrepresentation of taxa is currently occurring in *Alstroemeria*. Therefore, given that molecular data could reflect patterns of divergence more accordingly to the dynamic of local gene flow, an interesting approach is to evaluate taxonomic boundaries integrating both molecular and phenotypic data as potential taxonomic characters. Recent molecular and phenotypic integrated studies conducted in *Alstroemeria* complexes resulted beneficial when multiple sources of evidence are placed to solve questions about the integrity or the validity of previous taxonomic treatments. For example, when morphometric, cytogenetic and molecular data were employed in *A. hookeri* complex taxa [41, 67], all analyzed characters were partially consistent with the recognition of the new subspecies *Alstroemeria hookeri* subsp. *sansebastiana* [22], and supported the hypothesis of Muñoz & Moreira [14] of elevating *Alstroemeria hookeri* ssp. *cummingiana* to species level. Subsequent investigations were also conducted in other three complexes (*A. ligtu, A. magnifica* and *A. presliana*), eliciting similar evidence with significant taxonomic impact. In the *A. magnifica* complex, evidence from morphology, colorimetry and cytology support the change of the taxonomic status of *A. magnifica* var. *magenta.* Preliminary analyses based on chloroplast sequences (*rpl*32-*trn*L) also supported this observation, validating the separation of var. *magenta* from the other *A. magnifica* varieties (**Figure 11**). In the *A. ligtu* complex, a new entity was discovered based on cytogenetic data, and its taxonomic status was redefined [57, 58]. The molecular data, based on chloroplast DNA (*rpl*32-*trn*L region) support the separation of Coastal populations of *A. ligtu* subsp. *ligtu* from populations of the inland distribution range (**Figure 12**). Finally, in *A. presliana*, same chloroplast markers also confirm the lack of structure observed with AFLP data; nonetheless, both of them seem not concordant with previous studies conducted with phenotypic data [18, 30]. It is likely that different sources of divergence are shaping idiosyncratic processes of differentiation among species complexes of *Alstroemeria*, suggesting that a case by case evaluation might be required before reaching

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a consensus for a more genus-wide taxonomic perspective.

#### **5.3. Molecular markers in eliciting taxonomic status in** *Alstroemeria* **species complex**

The delimitation of species is a fundamental step for conducting natural and applied sciences, as they represent the main study unit for most areas of research (global evaluations of biodiversity, assessment and initiatives for biological conservation, etc.) [72]. In this sense, molecular approaches in taxonomy have been used under the assumption that observed

**Figure 10.** Maximum clade credibility tree (MCCT) inferred with the combination of chloroplast regions (trnL-S, rpl32-trnL, petA) for five species complexes of *Alstroemeria*, calculated with Bayesian inference criterion inferred with Mr. Bayes 3.2 [71]. Each tip represents an individual sampled per population and labels on branches depict posterior probabilities for each clade.

divergent patterns of genetic variation are the direct result of breaks in gene flow, leading to phenotypic and genotypic differences that sustain isolated and differentiated species and populations [69]. The simultaneous use of multiple molecular markers and criteria of delimitation has improved the taxonomic work, particularly helping to contextualize the role of microevolutionary processes in the species generation. As previously stated, species of *Alstroemeria* share attributes that could heavily influence patterns of micro evolutionary isolation and divergence, such as restricted seed and pollen dispersal and vegetative reproduction by rhizomes. Therefore, the wide distribution of taxonomic complexes in areas with contrasting topography and climatic conditions implies the existence of restrictions for gene flow, where substantial effects of ecogeographic isolation and divergence are expected in local diversification patterns [67]. Hence, micro evolutionary processes are currently underway and active [14], probably producing decoupled or unnoticeable patterns of divergence. Traditional taxonomic treatment in *Alstroemeria* has heavily relied on the interpretation of floral diversity and vegetative attributes, which has resulted in an important number of recognized taxa and species complexes described [14]. Nonetheless, because active of micro-evolutionary divergence may not ensure congruence among the diverse phenotypic and genotypic characters, it is likely that an under or overrepresentation of taxa is currently occurring in *Alstroemeria*. Therefore, given that molecular data could reflect patterns of divergence more accordingly to the dynamic of local gene flow, an interesting approach is to evaluate taxonomic boundaries integrating both molecular and phenotypic data as potential taxonomic characters. Recent molecular and phenotypic integrated studies conducted in *Alstroemeria* complexes resulted beneficial when multiple sources of evidence are placed to solve questions about the integrity or the validity of previous taxonomic treatments. For example, when morphometric, cytogenetic and molecular data were employed in *A. hookeri* complex taxa [41, 67], all analyzed characters were partially consistent with the recognition of the new subspecies *Alstroemeria hookeri* subsp. *sansebastiana* [22], and supported the hypothesis of Muñoz & Moreira [14] of elevating *Alstroemeria hookeri* ssp. *cummingiana* to species level. Subsequent investigations were also conducted in other three complexes (*A. ligtu, A. magnifica* and *A. presliana*), eliciting similar evidence with significant taxonomic impact. In the *A. magnifica* complex, evidence from morphology, colorimetry and cytology support the change of the taxonomic status of *A. magnifica* var. *magenta.* Preliminary analyses based on chloroplast sequences (*rpl*32-*trn*L) also supported this observation, validating the separation of var. *magenta* from the other *A. magnifica* varieties (**Figure 11**). In the *A. ligtu* complex, a new entity was discovered based on cytogenetic data, and its taxonomic status was redefined [57, 58]. The molecular data, based on chloroplast DNA (*rpl*32-*trn*L region) support the separation of Coastal populations of *A. ligtu* subsp. *ligtu* from populations of the inland distribution range (**Figure 12**). Finally, in *A. presliana*, same chloroplast markers also confirm the lack of structure observed with AFLP data; nonetheless, both of them seem not concordant with previous studies conducted with phenotypic data [18, 30]. It is likely that different sources of divergence are shaping idiosyncratic processes of differentiation among species complexes of *Alstroemeria*, suggesting that a case by case evaluation might be required before reaching a consensus for a more genus-wide taxonomic perspective.

**Figure 10.** Maximum clade credibility tree (MCCT) inferred with the combination of chloroplast regions (trnL-S, rpl32-trnL, petA) for five species complexes of *Alstroemeria*, calculated with Bayesian inference criterion inferred with Mr. Bayes 3.2 [71]. Each tip represents an individual sampled per population and labels on branches depict posterior

Despite the promising of these results, species from the Chilean group were mostly underrepresented, making difficult to obtain relevant evidence of local patterns of diversification, particularly to those depicting evolutionary trends or taxonomic integrity in species complexes. Nonetheless, while some progress has been achieved scrutinizing chloroplast sequences (*rpl*32-*trn*l), discordant results challenge the hypothesis integrity previously stated in several of these groups. While *Alstroemeria hookeri* and *A. presliana* complexes are retrieved as monophyletic clades, other groups like *A. magnifica* and *A. ligtu* are retrieved as

The delimitation of species is a fundamental step for conducting natural and applied sciences, as they represent the main study unit for most areas of research (global evaluations of biodiversity, assessment and initiatives for biological conservation, etc.) [72]. In this sense, molecular approaches in taxonomy have been used under the assumption that observed

**5.3. Molecular markers in eliciting taxonomic status in** *Alstroemeria* **species** 

probabilities for each clade.

paraphyletic (**Figure 10**).

248 Selected Studies in Biodiversity

**complex**

repetitive DNA has in the proportion of cut sites with restriction enzymes [17]. Since a more widespread consensus exist about the necessity of integrating different sources of molecular evidence and methodologies in species delimitation analyses [75], further work is required in the design of reliable and stable molecular markers for the study of

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With the onset of new and more accessible technologies for genome sequencing (Next Generation Sequencing or NGS), new possibilities have opened for the generation of more representative analyses of genetic diversity [76, 77]. Unfortunately, such techniques have been not widely implemented in *Alstroemeria*, except important breakthroughs like the sequencing and the annotation of the chloroplast genome in *A. aurea* [78]. The generation of single nuclear polymorphisms (SNP) in non-model organisms has been the approach of choice for high-throughput genome sequencing, adding improved genome representation and resolution for inter and intraspecific levels relationships [79]. The implementation of SNPs might result in a significant improvement in the estimation of genetic diversity and species limits in *Alstroemeria*, as SNPs represent codominant markers capable of providing a higher statistical power and an easier species comparability considering the available genomic resources compared to AFLP [80]. For taxonomic purposes, SNP could greatly improve the use of DNA barcodes to identify species through the use of specific DNA regions, especially when traditional approaches of taxonomy fail [81]. Obviously, the use of NGS and SNP techniques in *Alstroemeria* requires adjustments to overcome the limitations imposed by genome size and complexity, for which recent alternatives have been shown from the study of other equally complex organisms [82]. As such, the perspective of solving the taxonomic problems with molecular techniques in *Alstroemeria* remains promising, yet keeping in perspective its own limitations and challenges to reach the require tools to finally approach the inherent dynamics of macro and micro evolutionary patterns

Genetic divergence and population structure estimated with AFLPs and ISSR, have demonstrated the importance of molecular markers for conservation purposes in *Alstroemeria*. Integrative use of molecular data with other source of evidence (morphology, cytology and morphometry) give a best interpretation of lineage divergence with better argumentation for taxonomic delimitation in species complexes of *Alstroemeria*. Due to the high proportion of species complex in genus *Alstroemeria* in Chile, is necessary to carry out phylogenetic studies including the most infraspecifc taxa and more representative sampling, in addition with a major representation of the genome in the analyses. More efforts are needed in producing more stable molecular markers, in order to further implement integrative analyses. In this sense, it is likely that NGS will play a pivotal role helping to overcome present limitations of

natural species of *Alstroemeria*.

in this group.

**6. Concluding remarks**

molecular work in *Alstroemeria*.

**Figure 11.** Haplotype network inferred with trnL-F chloroplast spacer for individuals sampled from population of *A. magnifica* species complex. Network was constructed under parsimony criteria with TCS [73], as implemented in PopART [74].

#### **5.4. Perspectives and future work**

Previous studies have demonstrated that a consensus about the integrity of the taxa of *Alstroemeria* is far from being reached, as different patterns of differentiation may difficult to be elicited separately. In this sense, molecular markers have provided a natural framework to contextualize their evolutionary process, reconciling discordance observed from different character sources. Nonetheless, despite of their utility, molecular markers are not exempt of limitations that should be addressed in subsequent studies. One of the main limitations to reach a robust taxonomic hypothesis is the recurrent difficulty to obtain consistent molecular markers adaptable enough for interspecific and intraspecific analyses. These difficulties arise from the extraordinary large and complex genomic architecture of *Alstroemeria*, which is likely comprised of a large proportion of repetitive DNA (18–34 pg.) [17]. Our experience suggests that most nuclear markers tend to recurrently fail to retrieve single and readable copies through recurrent Sanger sequencing techniques, especially for the Internal Transcribed Spacer or ITS. Similarly, fragment analyses also exhibit levels of difficulties for consistent scoring, since the effect that

**Figure 12.** Haplotype network inferred with trnL-F chloroplast spacer for individuals sampled from population of *A. ligtu* species complex. Network was constructed under parsimony criteria with TCS [73], as implemented in PopART.

repetitive DNA has in the proportion of cut sites with restriction enzymes [17]. Since a more widespread consensus exist about the necessity of integrating different sources of molecular evidence and methodologies in species delimitation analyses [75], further work is required in the design of reliable and stable molecular markers for the study of natural species of *Alstroemeria*.

With the onset of new and more accessible technologies for genome sequencing (Next Generation Sequencing or NGS), new possibilities have opened for the generation of more representative analyses of genetic diversity [76, 77]. Unfortunately, such techniques have been not widely implemented in *Alstroemeria*, except important breakthroughs like the sequencing and the annotation of the chloroplast genome in *A. aurea* [78]. The generation of single nuclear polymorphisms (SNP) in non-model organisms has been the approach of choice for high-throughput genome sequencing, adding improved genome representation and resolution for inter and intraspecific levels relationships [79]. The implementation of SNPs might result in a significant improvement in the estimation of genetic diversity and species limits in *Alstroemeria*, as SNPs represent codominant markers capable of providing a higher statistical power and an easier species comparability considering the available genomic resources compared to AFLP [80]. For taxonomic purposes, SNP could greatly improve the use of DNA barcodes to identify species through the use of specific DNA regions, especially when traditional approaches of taxonomy fail [81]. Obviously, the use of NGS and SNP techniques in *Alstroemeria* requires adjustments to overcome the limitations imposed by genome size and complexity, for which recent alternatives have been shown from the study of other equally complex organisms [82]. As such, the perspective of solving the taxonomic problems with molecular techniques in *Alstroemeria* remains promising, yet keeping in perspective its own limitations and challenges to reach the require tools to finally approach the inherent dynamics of macro and micro evolutionary patterns in this group.
