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. 2023 May 20;131(6):1011–1023. doi: 10.1093/aob/mcad060

Intraspecific phylogeny of a Patagonian fescue: differentiation at molecular markers and morphological traits suggests hybridization at peripheral populations

V Guidalevich 1, N Nagahama 2, A S López 3, J P Angeli 4, P Marchelli 5,✉, M M Azpilicueta 6
PMCID: PMC10332399  PMID: 37209108

Abstract

Background and Aims

Grasses of the Festuca genus have complex phylogenetic relations due to morphological similarities among species and interspecific hybridization processes. Within Patagonian fescues, information concerning phylogenetic relationships is very scarce. In Festuca pallescens, a widely distributed species, the high phenotypic variability and the occurrence of interspecific hybridization preclude a clear identification of the populations. Given the relevance of natural rangelands for livestock production and their high degradation due to climate change, conservation actions are needed and knowledge about genetic variation is required.

Methods

To unravel the intraspecific phylogenetic relations and to detect genetic differences, we studied 21 populations of the species along its natural geographical distribution by coupling both molecular [internal transcribed spacer (ITS) and trnL-F markers] and morpho-anatomical analyses. Bayesian inference, maximum likelihood and maximum parsimony methods were applied to assemble a phylogenetic tree, including other native species. The morphological data set was analysed by discriminant and cluster analyses.

Key Results

The combined information of the Bayesian tree (ITS marker), the geographical distribution of haplotype variants (trnL-F marker) and the morpho-anatomical traits, distinguished populations located at the margins of the distribution. Some of the variants detected were shared with other sympatric species of fescues.

Conclusions

These results suggest the occurrence of hybridization processes between species of the genus at peripheral sites characterized by suboptimal conditions, which might be key to the survival of these populations.

Keywords: Intraspecific genetic differentiation, phylogenetic relationships, ITS, chloroplast DNA markers, morpho-anatomical analyses, plant traits, Festuca pallescens, Festuca, Patagonia, glacial refugia, hybridization, speciation

INTRODUCTION

Dryland ecosystems cover almost half of Earth’s land surface and house nearly 40 % of humanity, with outstanding importance for socioeconomic activities (Bradford et al., 2020; Maestre, 2021). Natural rangelands that develop in dryland areas constitute most of the world’s grasslands and are degraded due to overexploitation (Grau and Aide, 2008; James et al., 2013). In addition, plant communities of dryland ecosystems are highly sensitive to changes in temperature and precipitation regimes, such as those occurring due to global climate change (Magrin et al., 2014; Golodets et al., 2015). Increasing the knowledge of evolutionary processes in populations of key grassland species is necessary to both help predict upcoming changes and contribute to solutions.

Broadly distributed grassland species, covering environmental gradients and heterogeneous habitats, provide an interesting experimental design to study evolution and adaptation to suboptimal conditions (Maestre et al., 2012). Within these habitats, species have adapted in unique ways by developing deep and efficient root systems and water storage structures, or by going dormant during dry periods, to evade, avoid or resist drought (Davies et al., 2012; Moreno and Bertiller, 2015). These species tend to present large phenotypic and genetic variation (Manel et al., 2012). The level of genetic differentiation among central populations of the same species tends to be low due to historical persistence and the constant genetic exchange between individuals, but habitat heterogeneity could result in genetically distinct ecotypes (Hufford and Mazer, 2003; Eckert et al., 2008; Jakob et al., 2009). The continuous stress that populations undergo in dryland ecosystems may lead to adaptive changes and eventually even speciation processes (Hoffmann and Hercus, 2000). Natural hybridization may be a mechanism to promote adaptation, particularly in small and isolated populations that develop near the limits of their distribution range (Thompson et al., 2010). Specifically, glacial refugia during the Quaternary could have developed the conditions for the occurrence of ancient hybridization processes (Liu et al., 2018; Shepherd et al., 2022). Ultimately, hybridization may lead to adaptive evolution and speciation, which allows the new species to colonize extreme habitats to which neither of the parental species is adapted (Rieseberg et al., 2003; Liu et al., 2011). The recognition of ecotypes or hybrids can be complex, but the use of molecular markers complemented by the analysis of morphological character expression permits the reconstruction of phylogenetic trees (Pourebrahimi et al., 2022), thus making it possible to assign taxonomic status.

The Patagonian region constitutes the largest arid or semiarid ecosystem in southern South America. Climatic gradients mainly determined by the Andean mountains and a complex geomorphology give rise to a variety of environments and soil and vegetation types (Villalba et al., 2003; Gaitán et al., 2020). Plant communities in Patagonia are represented by two main functional groups: shrubs and perennial grasses (Bertiller and Bisigato, 1998). Natural rangelands dominated by tussock grasses and scattered shrubs are the main input for cattle raising and have been intensively used since the early 1900s (Defossé et al., 1990). Due to overgrazing and the effects of climate change, these valuable habitats undergo degradation and desertification, meaning that key species of grasses become less abundant due to a drastic reduction of their populations (Gaitán et al., 2017; Oliva et al., 2020). As perennial dominant grasses lose habitat, grasslands deteriorate and different species, particularly shrubs, may establish and dominate (Gonzalez and Ghermandi, 2021).

Festuca pallescens is a dominant allohexaploid species from natural rangelands of Patagonia (Dubcovsky and Martínez, 1992). This keystone species has a wide distribution and inhabits many diverse environments. It has been well studied and described morphologically and physiologically (Somlo et al., 1985; Bertiller et al., 1990; Fernández et al., 2004, 2006; Caballé et al., 2011; López et al., 2019, 2021), but information about its genetic variability is scarce (Dubcovsky and Martínez, 1991, 1992; López et al., 2020). Phenotypic variation among populations analysed in a common garden was observed and the molecular identification of a hybrid ecotype was carried out (López et al., 2018, 2020). This ecotype is found in a peripheral population clustered with another closely related polyploid, Festuca argentina, a sympatric species of F. pallescens (López et al., 2018). However, this previous study covered only part of the distribution range. Here, by extending the analysis to the whole natural range, we aim to analyse intraspecific variation and to reconstruct interspecific phylogenies involving other native species. Our main hypothesis is that the wide distribution range of this species, covering highly heterogeneous habitats, promoted genetic differentiation among populations. We also hypothesize that hybridization with sympatric species is possible in peripheral areas. We combined a molecular study of populations encompassing the whole distribution range with a thorough morphological analysis to describe intraspecific variation.

MATERIALS AND METHODS

Sampling strategy

Twenty-one populations of Festuca pallescens covering the distribution range of the species were analysed (Table 1, Fig. 1). Leaves were collected for DNA extraction and sequencing. Sampled individuals were identified in the field based on exomorphological characters described for the species (Nicora, 1978; Catalán and Müller, 2012). Additionally, four individuals of the sympatric hexaploid species Festuca gracillima, from the southernmost area of F. pallescens’ distribution, were collected and included in the analysis.

Table 1.

Geographic locations of the sampling sites of Festuca pallescens populations. GenBank accession numbers are included.

Population Abbreviation Sample site Latitude (south) Longitude (west) GenBank accession no.
ITS trnL-F
 1 ACA* Aguas Calientes, Neuquén, Argentina 36°41ʹ2″ 70°36ʹ37″ OP081817 KX701976
 2 LMI Los Miches, Neuquén, Argentina 37°6ʹ36″ 70°49ʹ3″ OP081817 KX701976
 3 HUI* Huinganco, Neuquén, Argentina 37°12ʹ17″ 70°37ʹ12″ OP081817 KX701976
 4 CHO Chos Malal, Neuquén, Argentina 37°21ʹ38″ 70°7ʹ23″ OP081817 KX701976
 5 CLI* Catan Lil 2, Neuquén, Argentina 39°45ʹ40″ 70°37ʹ7″ KX688222 KX701976
OP081824
 6 SRA San Ramón Ranch, Río Negro, Argentina 41°10ʹ32″ 70°59ʹ1″ KX688222 KX701976
 7 PIL* Pilcaniyeu Experimental Field, Río Negro, Argentina 41°3ʹ40″ 70°31ʹ1″ KX688222 KX701976
 8 MON Montoso, Chubut, Argentina 42°42ʹ52″ 71°01ʹ59″ KX688222 KX701976
 9 JAC Ingeniero Jacobacci, Río Negro, Argentina 41°55ʹ9″ 69°12ʹ58″ KX688222 KX701976
KX701978
 10 SOM Somuncura Plateau, Río Negro, Argentina 41°25ʹ1″ 66°58ʹ1″ KX688222 KX701976
 11 YAG Yague, Chubut, Argentina 42°57ʹ0″ 71°12ʹ0″ KX688222 KX701976
OP081824
 12 APE* Arroyo Pescado, Chubut, Argentina 43°2ʹ49″ 70°58ʹ2″ KX688222 KX701976
 13 CRO* Cronómetro, Chubut, Argentina 43°14ʹ19″ 71°4ʹ54″ OP081820 KX701976
 14 GOC Gobernador Costa, Chubut, Argentina 44°1ʹ12″ 70°51ʹ36″ KX688222 KX701976
 15 RPI Río Pico, Chubut, Argentina 44°8ʹ24″ 71°26ʹ24″ KX688222 KX701976
 16 FON* Fontana, Chubut, Argentina 44°56ʹ46″ 71°31ʹ09″ OP081819 KX701976
 17 RMA* Río Mayo, Chubut, Argentina 45°28ʹ12″ 69°49ʹ48″ KX688222 KX701976
OP081816 OP081821
OP081822
OP081824
 18 LBA* Mte Lago Bs As, Santa Cruz, Argentina 46°36ʹ0″ 71°7ʹ48″ KX688222 KX701976
 19 LCO Laguna Colorada, Santa Cruz, Argentina 51°40ʹ15″ 69°53ʹ58″ KX688222 KX701976
 20 BLE* Bajo la Leona, Santa Cruz, Argentina 51°31ʹ48″ 69°42ʹ4″ KX688222 OP081823
 21 GAI* Guer Aike, Santa Cruz, Argentina 51°37ʹ33″ 69°37ʹ53″ OP081818 OP081825
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Fig. 1.

Fig. 1.

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(A) Geographic distribution of the ITS variants of the evaluated populations of F. pallescens. Festuca pallescens variants are coloured in shades of green. (B) Bayesian tree based on nuclear ITS markers. Numbers above the branches indicate posterior probability values. The scale bar shows the expected substitutions per site. Pappostipa speciosa var. speciosa and Poa ligularis were used as outgroups.

DNA extraction and PCR amplification

DNA from at least three individuals per population was extracted. Leaf tissue was frozen using liquid nitrogen and ground to fine powder with an automatic mixer mill (Resch, Germany). DNA extraction followed the protocol of Doyle and Doyle (1987), with slight modifications (Gonzalo-Turpin and Hazard, 2009). Two DNA markers were used for the phylogenetic analysis: the internal transcribed spacer (ITS) and a chloroplast DNA region (trnL-F). The complete ITS region (ITS1-5.8S-ITS2) was amplified using the primers CY1-CY3 (Wright et al., 2006), ~600–700 bp in length (Torrecilla and Catalán, 2002; Catalán et al., 2004). For the amplification we used 40 ng of DNA as template, 0.625 U of GoTaq DNA polymerase (Promega, Madison, WI, USA) with 1× Colorless GoTaq® reaction buffer (Promega), 1.5 mm of MgCl2, 0.25 mm of each dNTP and 0.3 µm of each primer in a total volume of 30 µL. PCR reactions were carried out following this program: 4:30 min at 95 °C, 30 cycles of 30 s at 94 °C, 1 min at 56 °C and 2 min at 72 °C, and a final extension of 10 min at 72 °C. The trnL-F region (~1000 bp, Catalán et al., 2004) was amplified using the universal primers c and f (Taberlet et al., 1991) with 100 ng of DNA, 1 U of GoTaq DNA polymerase (Promega, Madison, WI, USA) with 1× Colorless GoTaq® reaction buffer (Promega), 2 mm MgCl2, 0.2 mm of each dNTP and 0.2 µm of each primer in a total volume of 50 µL. The amplification program used for this marker was as follows: 1 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C, followed by a final extension of 7 min at 72 °C. PCR products for both markers were checked for positive amplification in 1.5 % agarose gels, stained with Gel Red, and visualized with a UV transilluminator. Amplified regions yielded bands of ~700 bp (ITS) and 1000 bp (trnL-F), which were purified using the ExoSAP-IT™ PCR Product Cleanup (Thermo Fisher) commercial kit and then sequenced in a capillary sequencer (ABI 3700, Unidad de Genómica, Instituto de Biotecnología de INTA, Hurlingham, Argentina).

Phylogenetic analyses

The ITS sequences were aligned using Muscle with manual adjustments when needed in AliView 1.26 (Larsson, 2014). For the phylogenetic analysis of the ITS region we additionally used sequences from 14 Patagonian species of Festuca retrieved from GenBank (Supplementary Data Table S1). In addition, a sequence from the ecotype of F. argentina previously mentioned was also included (López et al., 2018). Two Patagonian grasses (Poa ligularis var. ligularis and Pappostipa speciosa var. speciosa) were used as outgroups. All the ITS and trnL-F sequences of F. pallescens and F. gracillima were deposited in GenBank (accession numbers are provided in Supplementary Data Table S1).

A matrix of 24 sequences of 578 characters each was generated for the analysis of the ITS region. Gaps were treated as missing data and indels as point mutations. Boundaries of each sequence were established by alignment with the species of Festuca retrieved from GenBank (14 species). To assess the phylogenetic relationships between species of Patagonia and among populations of F. pallescens we performed analyses based on Bayesian inference (BI), maximum parsimony (MP) and maximum likelihood (ML) criteria. The IB, MP and ML analyses were carried out with MrBayes 3.2.7a (Ronquist et al., 2012), TNT 1.5 (Goloboff and Catalano, 2016) and the online software RAxML (Kozlov et al., 2019), respectively. We used JModelTest 2.1.10 to establish the best-fitting nucleotide substitution model that is required to run BI and ML analyses (Darriba et al., 2012). This resulted in model GTR + G according to both the Akaike information criterion (AIC) and Bayesian information criterion (BIC). Bayesian analyses were carried out by running pre-established parameters (1 million generations initiated from different random trees, sampling every 100th generation model parameters such as nucleotide substitution rates, γ shape, proportion of invariable sites, nucleotide frequency) estimated by MrBayes. After burn-in (discarding 25 % of the total sampling), parameters were sampled when reaching stationarity. The ITS consensus tree, obtained from 15 000 trees, was supported by branch values from the posterior probability. Clades with >95 % posterior probability values are considered well supported. Parsimony analyses were executed by subjecting the data set to a heuristic search strategy to find all equally parsimonious trees (MULPARSon, TBR) with 10 000 replicates, saving no more than ten trees of length equal to or shorter than 10 (López et al., 2018). These saved trees were collapsed to a consensus tree that was used as a negative constraint for a second search. All parsimonious trees obtained were used to compute a strict and 50 % majority rule consensus tree. Bootstrap support for branches was calculated through heuristic searches of 1000 replicates. Clades with bootstrap values <50 are shown unresolved. We used the software FigTree (http://tree.bio.ed.ac.uk/software/figtree/) to edit and visualize the consensus tree. To reconstruct the relationships between haplotypes we ran the program Network 10.2.0.0, which works with a median-joining network algorithm (Bandelt et al., 1999). The dataset used for the reconstruction contained the chloroplast DNA sequences of F. pallescens (seven haplotypes), F. gracillima, F. purpurascens and F. argentina and the sequence corresponding to the hybrid ecotype (López et al., 2018).

Morphological data

Twelve populations of F. pallescens that differ in either of the two evaluated molecular markers were selected for morphological characterization (Table 1). Plant material was obtained from the living collection of F. pallescens populations held at the Campo Experimental Agroforestal Trevelin (CEAT INTA Esquel; 43°07ʹ30″S, 71°33ʹ04″W, 356 masl), Argentina. Twenty-four quantitative and nine qualitative morphological traits were measured in 6–12 specimens of each population (Supplementary Data Table S2). Morphological characters included in this study are those traditionally used for the delimitation of species in Festuca (Dubcovsky and Martínez, 1988; Catalán and Müller, 2012). In this genus, the distribution of sclerenchyma in the leaf cross-section is a main trait for species identification (Aiken et al., 1985; Dubcovsky and Martínez, 1988). Therefore, leaf anatomy characters were observed in the middle region of the blade of the penultimate leaf of the short internodes zone in all specimens studied. The identification of different combinations of adaxial and/or abaxial girders of sclerenchyma of vascular bundles was based on leaf cross-sections stained with safranin.

Statistical analysis

The quantitative variables were tested for normality with the Shapiro–Wilks test (Mahibbur and Govindarajulu, 1997), as well as using diagram boxes. Homogeneity of variance was checked with Bartlett’s test. One-way ANOVA (α = 0.05) was performed to evaluate the significance of the differences among the specimens for each trait. Tukey’s test (P < 0.05) was applied for a posteriori comparison of each pair of means. For the analysis of the variable lemma awn length, population ACA (1) was excluded because plants lack awns in the lemma (see Results section).

The morphological data set (Supplementary Data Table S3) was analysed by discriminant and cluster analyses, with each character represented by mean values. Morphological character values were standardized prior to use in multivariate analyses. Pearson and Spearman correlation coefficients were estimated to identify pairs of highly correlated characters that may distort multivariate analyses (Michener and Sokal, 1957; Conover, 1999). Discriminant analysis allows samples to be classified within predefined groups using the discriminant functions adjusted for maximizing the between-groups to within-groups ratio of variance. Cluster analysis was performed to reveal the structure residing in the morphological dataset. Sample similarities using the morphological data matrix were calculated based on the Euclidian distance, and the average linkage hierarchical agglomerative method was used to establish clusters. The statistical analyses were performed using Infostat v. 2015 (Di Rienzo et al., 2015).

RESULTS

Variation at the ITS region

Six variants were identified at the ITS region along the distribution range of F. pallescens. The most common variant (F.pallescensVar1) was detected in 14 populations. Four northern populations [ACA (1), LMI (2), CHO (3) and HUI (4)] shared a distinctive ITS sequence, and the other four variants were found in two western populations [CRO (13) and FON (16)], one eastern population [RMA (17)], and one southern population [GAI (21)] (Fig. 1A). The ITS dataset included 19 taxa: F. pallescens (with six variants), 16 native species of Festuca, including the hybrid ecotype of F. argentina (Supplementary Data Table S1), and two outgroups (P. ligularis and P. speciosa). From the 578 aligned nucleotide positions, 73 (12.63 %) were parsimony-informative. The three phylogenetic analyses exhibited similar topologies, with two major clades that separate the F. pallescens variants from the hybrid ecotype. The BI tree showed the highest support values (Fig. 1A); MP and ML can be found in Supplementary Data Fig. S1. The ITS variants found in F. pallescens specimens formed a well-supported clade, associated with F. gracillima. A strong association with other Patagonian native species (e.g. F. purpurascens) and with northern species (e.g. F. ventanicola) was also observed. In a separate clade, the hybrid ecotype variant clustered together with F. argentina. This clade is sister to only one species distributed in the extreme north of Argentina (F. superba).

Variation in the chloroplast DNA (trnL-F) region

A total of seven chloroplast haplotypes were found among the analysed populations. The topology of the network shows the ancestral character of the most frequent haplotype (Hp1) (Fig. 2). Haplotype 4 shares the same six-nucleotide insertion as F. purpurascens but differs by two nucleotides in other positions. Haplotypes 2 and 5 share an insertion of 22 nucleotides but differ at two positions. One of these polymorphisms is an ambiguity (W in IUPAC code), with Hp2 showing W, and two alternative nucleotides observed in Hp1, Hp5, Hp6 and Hp7 (T), and Hp3 and Hp4 (A). Haplotype 7 shows high similarity with F. gracillima’s haplotype and is distanced from Hp1 by nine mutations. Additionally, the sympatric species F. argentina differs from Hp1 by 13 mutated positions and shares the same haplotype as the hybrid ecotype (Fig. 2).

Fig. 2.

Fig. 2.

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Geographic distribution of the haplotypes detected in F. pallescens populations along the entire distribution range. Putative glacial refugia [lowland, peripheral and valley refugia as described by Sérsic et al. (2011)] for different plant species are delimited with blue lines. The box on the right shows the haplotype network. Haplotypes are represented with different-sized circles according to their frequencies (HyEc, hybrid ecotype; Fgra, F. gracillima; Fpur, F. purpurascens; Farg, F. argentina). Point mutations are shown as black lines and indels as red lines.

The most frequent haplotype (Hp1) was found in 18 populations (Fig. 2). Haplotypes were fixed in 17 populations, 15 exhibiting the common haplotype and the other two [BLE (20) and GAI (21)] exclusive types. Intrapopulation variation was observed in four populations [CLI (5), JAC (9), YAG (11) and RMA (17)] (Fig. 2).

Morphological analyses

Significant differences between two or more populations for 20 of the 24 continuous variables were detected (ANOVA; P < 0.05). The average values and standard deviations of the quantitative traits analysed, as well as the results of a posteriori tests, are summarized in Supplementary Data Table S2. Population ACA (1), from northern Patagonia, was clearly the most differentiated. Its specimens showed ciliated ligules with significantly shorter hairs (P < 0.05). Northern populations [ACA (1), CLI (5), PIL (7), HUI (4) and JAC (9)] presented shorter hairs in the abaxial surface of leaves compared with southern populations [APE (12), CRO (13), FON (16), RMA (17), LBA (18), BLE (20) and GAI (21)]. Specimens of populations ACA (1) and HUI (4) presented longer ligules and were different compared with other populations (P < 0.05). In addition, populations ACA (1), HUI (4), CLI (5) and JAC (9) showed significantly shorter spikelets (P < 0.0001). Two populations of south Patagonia [LBA (20) and GAI (21)] had longer synflorescences (P < 0.0001). Plants from the CRO (13) and FON (16) populations presented longer lodicules (P < 0.0001).

Most variables were not correlated, except for spikelet length with lemma length (Pearson = 0.83), spikelet length with paleae length (Pearson = 0.87), and lemma length with paleae length (Pearson = 0.9). Pearson and Spearman correlation values were similar. Since the use of highly correlated characters is an implicit weighting of these characters and suggests potential multicollinearity problems, the total number of variables was reduced to 30 for final analyses (Supplementary Data Table S3).

Discriminant analysis led to the identification of morphological patterns that allowed a good discrimination among some populations. Populations ACA (1) and HUI (4) were clearly discriminated when the first two discriminant functions (axis 1 and axis 2) were plotted (Fig. 3), while ACA (1), HUI (4) and CLI (5) were discriminated considering the axes 1 and 3 (Supplementary Data Fig. S2). The first three axes accounted for 62.82 % of the variation. The absolute values of the coefficients of the standardized discriminant functions are shown in Supplementary Data Table S3.

Fig. 3.

Fig. 3.

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Scatterplot of scores derived from discriminant functions axis 1 and axis 2 produced by discriminant analysis applied to 30 morphological variables for the 12 populations of Festuca pallescens studied: ACA (solid squares), CLI (solid inverted triangles), PIL (solid diamonds), HUI (solid triangles), JAC (solid circles), CRO (open triangles), APE (open inverted triangles), GAI (open diamonds), LBA (open squares), RMA (open circles), FON (open pentagon), and BLE (open hourglasses).

Leaf cross-sections revealed the presence of sclerenchyma under the abaxial epidermis and in the ribs under the adaxial epidermis in all specimens analysed. In addition, different combinations of adaxial and/or abaxial girders of sclerenchyma in vascular bundles were observed, depending on the population (Fig. 4). Population ACA (1) was the only one with double girders of sclerenchyma (adaxial to abaxial) in the main and secondary vascular bundles. In other populations the main and secondary vascular bundles showed different combinations with presence and/or absence of abaxial girders of sclerenchyma. In population ACA (1) all specimens showed abaxial girders of sclerenchyma in the main vascular bundle and 50 % of them presented double girders. In secondary vascular bundles all plants also presented double girders of sclerenchyma. Populations that presented abaxial girders of sclerenchyma in the main vascular bundle were HUI (4), CLI (5), PIL (7), JAC (9), APE (12), CRO (13), RMA (17) and GAI (21) (see different percentages in Fig. 5). In populations FON (16), LBA (18) and BLE (20), girders of sclerenchyma in the main vascular bundle were not observed. Populations that presented abaxial girders of sclerenchyma in secondary vascular bundles were PIL (7), JAC (9), APE (12), CRO (13) and GAI (21) (see percentages in Fig. 5). In populations HUI (4), CLI (5), FON (16), RMA (17), LBA (18) and BLE (20), girders of sclerenchyma in secondary vascular bundles were not present.

Fig. 4.

Fig. 4.

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Leaf cross-section of specimens stained with safranin showing examples of the different combinations of adaxial and/or abaxial girders of sclerenchyma of vascular bundles. Population names follow those of Supplementary Data Table S1. (A) Adaxial to abaxial girders of sclerenchyma in main vascular bundle. (B) Adaxial to abaxial girders of sclerenchyma in secondary vascular bundles. (C) Abaxial girders of sclerenchyma in main vascular bundle. (D) Abaxial girders of sclerenchyma in secondary vascular bundles. (E) Secondary vascular bundles without girders of sclerenchyma. (F) Main vascular bundle without girders of sclerenchyma.

Fig. 5.

Fig. 5.

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Dendrogram resulting from cluster analysis obtained with the morphological data of F. pallescens populations. The dendrogram includes morpho-anatomical results of leaf cross-sections (following Fig. 4) and molecular variation according to ITS and cpDNA. The scale corresponds to Euclidean distance.

In the cluster analysis, populations were grouped in terms of their similarity according to the 30 morphological traits analysed (Supplementary Data Table S3). Considering a reference line equal to 50 % of the maximum distance (a frequently used criterion) to validate clusters, the cut-off line was established at distance 5.83. The dendrogram shows that population ACA (1) is clearly separated from the rest and populations of north and south Patagonia were grouped into two main clusters (Fig. 5).

DISCUSSION

Maintaining genetic diversity across populations of a species is essential to ensure its survival, particularly under the current climate change context. We took the first steps towards describing differentiation among populations of a widespread keystone species of an arid and semiarid region. In this study, we found intraspecific variation at molecular markers and morpho-anatomical traits. Edge populations of F. pallescens hold different genetic variants in comparison with those located at the centre of the distribution. Distinct populations can be observed particularly at northern, eastern and southern sites. In general, the same pattern is observed for both markers. Similarly, morphological traits also separated populations into two latitudinal groups.

Intraspecific phylogeny

The Poaceae family includes >700 genera of economic and ecological significance, but phylogenetic relationships among species are complex and sometimes unresolved (Grass Phylogeny Working Group, 2001; Soreng et al., 2017). In addition, phylogenetic studies encompassing the relationships among Patagonian fescues are limited (Dubcovsky and Martínez, 1988; Ospina González, 2016; López et al., 2018). The genus Festuca holds >500 species all over the world (Inda et al., 2008). Although well supported phylogenies exist, the representation of Patagonian species is very scarce, e.g. only F. argentina, F. subantarctica, F. purpurascens, F. gracillima, F. magellanica and F. pyrogea were included (Torrecilla and Catalán, 2002; Torrecilla et al., 2003; Catalán et al., 2004; Inda et al., 2008; Minaya et al., 2017). In a previous study, we reported the position of F. pallescens within these phylogenies, clearly positioned within the ‘fine-leaved lineage’ (López et al., 2018). Here, with an enlarged sampling, we reconstructed the relationships between the Patagonian fescue species and the F. pallescens variants. Our results show that most of F. pallescens’ variants are closely related to F. gracillima, a sympatric species that occurs at the southernmost end of the distribution. This species is described as a complex together with F. pallescens (Dubcovsky and Martínez, 1991; Torrecilla and Catalán, 2002; Catalán and Müller, 2012). The variant detected in the four northernmost populations is separated from the main clade, forming an unresolved polytomy to another sympatric species, F. purpurascens. The natural distribution of F. purpurascens overlaps with F. pallescens’ distribution, and the two species showed high genetic similarities at both molecular markers.

Festuca gracillima, F. purpurascens and all the variants of F. pallescens were grouped with most species of Festuca, and were separated, with strong support, from a group that included F. argentina, F. superba and the hybrid ecotype. These results are consistent with Catalán et al. (2004), in which the festucoid grasses are separated into two major linages: ‘broad-leaved’ and ‘fine-leaved’ (Inda et al., 2014). Festuca argentina would belong to the first group, while F. purpurascens and F. pallescens would presumably belong to the latter (Catalán et al., 2004; Inda et al., 2008; López et al., 2018). In a further study of molecular, anatomical and micro-morphological characters, these species are also separated in different groups (Ospina González, 2016), but F. argentina is placed in an ambiguous position, close to either broad- or fine-leaved lineages. Even though Ospina González (2016) placed F. purpurascens and F. pallescens in a fine-leaved group according to his molecular results, anatomically he describes F. purpurascens within the broad-leaved lineage while F. pallescens is placed in the fine-leaved lineage together with F. gracillima. Clearly the high variation within and among species of the Festuca genus deserves more attention and further studies will contribute to a better comprehension of the relationships of the species belonging to this genus.

Genetic differentiation along the natural range

We identified eight distinct populations that differed from the most frequent ITS variant. Four of these populations are located at the northern edge of the distribution, and all the individuals sampled share the same sequence. This region was affected by sea introgressions and glaciations during the Miocene and Pleistocene (Ramos, 1982; Folguera and Ramos, 2002; Rabassa et al., 2005), which makes it a perfect spot for stochastic factors to develop differentiation from the central populations (Eckert et al., 2008). Within this northern region the extent of glaciations was limited to the foothills of the Andes (Flint and Fidalgo, 1964; Hollin and Schilling, 1981), leaving many ice-free areas where species could survive in situ (e.g. Markgraf et al., 1995; Sérsic et al., 2011; Soliani et al., 2012). At this latitude, F. pallescens shares its habitat with other native species of the genus, which are morphologically similar (e.g. F. acanthophylla var. acanthophylla and F. acanthophylla var. scabriuscula). Marginal populations are considered active regions for speciation processes which are particularly common in plants (Crawford, 2010; Rajakaruna, 2018). Ancient hybridization and speciation processes at glacial refugia might be a possible explanation of the variants shared by northern populations, but also of the southern variants found in this study (see next section).

Four populations at the species range edges have shown distinct variants. The occurrence of differentiation at suboptimal locations could imply an evolutionary advantage under extreme climatic conditions, allowing the species to colonize and remain at these further frontiers. For instance, plant species increase their ploidy level with latitude (Rice et al., 2019) and most Patagonian fescues are mainly polyploids (Dubcovsky and Martínez, 1988, 1992). Polyploidy diversifies an organism’s genetic background and provides selective advantages in novel or stressful environments (Van de Peer et al., 2017), and can also be a common way of sympatric speciation through hybridization processes (Wei et al., 2019; Gordon et al., 2020). Therefore, showing distinct genetic and morpho-anatomical variants, edge populations of F. pallescens could either suggest an ecological advantage (i.e. drought or salinity stress) or reinforce the possibility of hybridization with sympatric species.

A similar pattern of distribution of the genetic variation was displayed at the chloroplast level. The less frequent haplotypes found in our study were detected in marginal populations (east and south of the distribution). The populations carrying the alternative variants match the distribution of different putative glacial refugia described for Patagonian plant species (Cosacov et al., 2010; Sérsic et al., 2011; Baranzelli et al., 2022) (Fig. 2). Two haplotypes found in the southernmost populations near one of the aforementioned refugia (Sérsic et al., 2011) are of particular interest: Hp4 and Hp7 from populations BLE (20) and GAI (21), respectively. The haplotype 4 shares a step mutation with one haplotype found in population RMA (17), closer to the centre of the natural range for the species, but it also presents a six-nucleotide insertion that matches the sequence of F. purpurascens. On the other hand, Hp7 is almost an exact match of F. gracillima’s sequence. The populations BLE (20) and GAI (21) belong to the southernmost region of the distribution, where it overlaps with the distribution range of F. purpurascens and F. gracillima (Dubcovsky and Martínez, 1988; Ospina González et al., 2015). Hybridization with other species of the genus that share the same ploidy count is plausible and it has been argued as either a cause or consequence of speciation (Dubcovsky and Martínez, 1991, 1992; Oliva, 1996; Šmarda and Stančík, 2005; Ospina González, 2016; Fernández et al., 2017). Fescues are known to hybridize within the genus (Humphreys et al., 1995; Inda et al., 2014), and a putative hybrid ecotype has already been described in a location with suboptimal conditions and isolated from the centre of the distribution (López et al., 2018). Interspecific hybrids have also been described as common among other species of the Poaceae, and these events can complicate the reconstruction of phylogenetic relations between species (Díaz-Pérez et al., 2014; Baiakhmetov et al., 2020).

The high genetic diversity detected in population RMA (17) at both markers deserves attention. This population is close to a described plant refuge and a phylogeographical break for several species reviewed by Sérsic et al. (2011). The steppe vegetation is poorly represented since most studies were devoted to temperate forest species. However, in situ survival throughout glacial cycles was suggested based on phylogeography and ecological niche modelling that indicated stable geographical distribution of two Hordeum species for at least the Holocene (Jakob et al., 2009). Past vegetation reconstruction within the southern distribution range of F. pallescens confirms its presence during the Holocene (Echeverría et al., 2022). The legacy of the glacial era is still imprinted in the current taxa, but F. pallescens was never studied. Our results are the first information contributing to knowledge of the evolutionary history of the species. Future studies should include other chloroplast markers to infer phylogeographical patterns.

Morphological and anatomical comparisons

In the genus Festuca there are vegetative characters that are of great taxonomic importance and one of them is the internal morphology of the leaves (Saint-Yves, 1927; Parodi, 1953; Aiken et al., 1985). In Patagonian fescues, the distribution of sclerenchyma on the leaves not only helps to explain the geographical distribution of some of the species but also taxonomically separates the species into different groups (Dubcovsky and Martínez, 1988). Festuca pallescens can be distinguished by the presence of sclerenchyma under the abaxial epidermis and in the ribs under the adaxial epidermis, but does not possess girders of sclerenchyma in the main and secondary vascular bundles (Dubcovsky and Martínez, 1988). The morphological analyses allowed us to confirm the taxonomic identity of the studied specimens and, in addition, to detect the phenotypic variability of the species throughout its natural distribution range.

The specimens from ACA population (1) were morpho-anatomically the most different among all populations analysed. Unlike the other populations, plants in ACA presented awnless lemmas, double girders of sclerenchyma (adaxial to abaxial) in the main and secondary vascular bundles and ligules with the shortest hairs. According to Dubcovsky and Martínez (1988), these characteristics are observed in F. purpurascens, F. argentina, F. acanthophylla var. acanthophylla, F. cirrosa, F. monticola and F. acanthophylla var. scabriuscula. Within the region of the ACA population, F. pallescens grows in sympatry with F. acanthophylla var. scabriuscula and var. acanthophylla. The specimens of the ACA population presented other leaf anatomical traits (e.g. number of vascular bundles and the arrangement of subepidermal sclerenchyma) that are more similar to those typical of these two species than to F. pallescens. Similarly, the southernmost populations [BLE (20) and GAI (21)] live in sympatry with F. gracillima. Although leaf anatomy in BLE specimens is similar to that in F. gracillima, that in GAI is not, because they present abaxial girders in the main and secondary vascular bundles. As previously stated for genetic variability, these results might be evidence of ancient hybridization processes that occurred during the Pleistocene at periglacial refugia at both the northern and southern limits of the distribution range. Different putative glacial refugia have been described within the current distribution range of F. pallescens, from north-west Patagonia (~35° S) to the south of Santa Cruz (~51° S) (Sérsic et al., 2011). Moreover, the distribution ranges of F. acanthophylla and F. purpurascens in the north and F. gracillima in the south match with some of these areas (Dubcovsky and Martínez, 1988) (Fig. 2). The coexistence of these species with F. pallescens in confined proximity and through many recolonization cycles could have promoted interspecific crossing. Hybridization during prolonged isolation in glacial times was reported for other species around the world (e.g. Consaul et al., 2010; Klein and Kadereit, 2016; Liu et al., 2018; Shepherd et al., 2022) and also in Patagonia (e.g. Tremetsberger et al., 2009; Soliani et al., 2012; Azpilicueta et al., 2014).

A latitudinal pattern in the distribution of genetic variation is frequently found within Patagonian species, both plants and animals, mainly related to historical processes like glaciations (e.g. Bekessy et al., 2002; Marchelli and Gallo, 2006; Azpilicueta et al., 2009, 2014; Arana et al., 2010; Cosacov et al., 2010; Lessa et al., 2010; Mathiasen and Premoli, 2010; Sérsic et al., 2011; Soliani et al., 2012, 2015; Vera-Escalona et al., 2012; Baranzelli, 2017; Fasanella et al., 2017; Renny, 2022). The variation detected in our study indicates that F. pallescens would follow the same latitudinal pattern. Northern populations shared some traits that differed from those recorded in the southern populations. For example, the ACA (1) and HUI (4) populations presented the longest ligules. In addition, the populations of northern Patagonia [ACA, HUI, CLI (5), PIL (7) and JAC (9)] differed from the other populations by presenting significantly shorter hairs on the abaxial surface of leaves. Moreover, the ACA, HUI, CLI and JAC populations presented significantly shorter spikelets. Discriminant analyses separated the ACA, HUI and CLI populations, leaving the rest of the populations without a defined grouping pattern. From the results of the cluster analysis, it is clear that the ACA population is separate from the rest of the populations, which grouped into two main subgroups according to their latitudinal geographical distribution (north and south populations).

Conclusions

Our results showed a low genetic differentiation among central populations but distinctiveness at peripheral sites, suggesting that hybridization of F. pallescens with sympatric species is possible; morphological traits and molecular markers converged on similar outcomes. Low genetic variability is an expected outcome of permanent persistence in situ as suggested by Jakob et al. (2009) for Hordeum spp. Pollen and phytolith records showed evidence of the presence of current steppe species belonging to the Poaceae family during the early Quaternary, reinforcing this possibility (Palazzesi and Barreda, 2012; Palazzesi et al., 2021). In the north, the group of analysed populations shared the same distinct molecular variants but displayed differences in morphological traits. Hybridization with sympatric species at these latitudes may be suggested, and even different stages of speciation can be proposed given the high separation of the ACA population from the rest of the northern populations. This population is at the limit of the distribution range. A similar situation can be inferred at the southern edge, where populations resemble those of F. gracillima in both molecular and morphological characters. Marginal sites usually offer suboptimal conditions for a species; therefore, hybridization can play a key role in adaptation (Španiel and Rešetnik, 2022).

Supplementary Material

mcad060_suppl_Supplementary_Material
Click here for additional data file. (411.6KB, docx)

ACKNOWLEDGEMENTS

We are grateful to A. Beider, A. Mogni, E. Vivar, G. Oliva, G. Siffredi, H. Morgana, M. Easdale and W. Opazo for helping with the collection of biological material used for this study. We specially thank F. Umaña for the assistance in the use of QGIS for the preparation of Figs 1 and 2, and R. Vidal Russell for the assistance in the use of TNT software. Sampling permissions were provided by the corresponding provinces (Neuquén, Río Negro, Chubut and Santa Cruz, Argentina).

Contributor Information

V Guidalevich, INTA Bariloche – IFAB (INTA-CONICET), Modesta Victoria 4450, 8400, Bariloche, Argentina.

N Nagahama, EEAf Esquel INTA, Chacabuco 513, 9200, Esquel, Argentina.

A S López, INTA Bariloche – IFAB (INTA-CONICET), Modesta Victoria 4450, 8400, Bariloche, Argentina.

J P Angeli, EEAf Esquel INTA, Chacabuco 513, 9200, Esquel, Argentina.

P Marchelli, INTA Bariloche – IFAB (INTA-CONICET), Modesta Victoria 4450, 8400, Bariloche, Argentina.

M M Azpilicueta, INTA Bariloche – IFAB (INTA-CONICET), Modesta Victoria 4450, 8400, Bariloche, Argentina.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: species and GenBank accession numbers. Table S2: one-way ANOVA. Table S3: discriminant analysis. Figure S1: maximum parsimony and likelihood tree based on nuclear data set. Figure S2: scatterplot of scores derived from axis 1 versus axis 3 produced by discriminant analysis applied to 30 morphological variables for the 12 populations studied.

FUNDING

This study was financed by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación through PICT 2017-2379, Instituto Nacional de Tecnología Agropecuaria through PE I142 and Consejo Nacional de Investigaciones Científicas y Técnicas through PUE CONICET 069. V.G. was supported by a fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas and J.P.A. by a cofinanced fellowship from Instituto Nacional de Tecnología Agropecuaria and Consejo Nacional de Investigaciones Científicas y Técnicas.

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