RESEARCH ARTICLE
Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes David S. Gernandt1,8, Xitlali Aguirre Dugua1, Alejandra Vázquez-Lobo2, Ann Willyard3, Alejandra Moreno Letelier4, Jorge A. Pérez de la Rosa5, Daniel Piñero6, and Aaron Liston7
Manuscript received 19 October 2017; revision accepted 24 January 2018. 1 Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico 2 Centro de Investigación en Biodiversidad y Conservación, Universidad Autónoma del Estado de Morelos, Col. Chamilpa, Cuernavaca, Morelos 62209, Mexico 3 Biology Department, Hendrix College, Conway, Arkansas 72032, USA 4 Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico 5 Centro Universitario de Ciencias Biológicas y Agropecuarias, Instituto de Botánica, Universidad de Guadalajara, Nextipac, Zapopan, Jalisco 45510, Mexico 6 Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico 7 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA 8
Author for correspondence (e-mail:
[email protected])
Citation: Gernandt, D. S., X. Aguirre Dugua, A. Vázquez-Lobo, A. Willyard, A. Moreno Letelier, J. A. Pérez de la Rosa, D. Piñero, and A. Liston. 2018. Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes. American Journal of Botany 105(4): 1–15. doi:10.1002/ajb2.1052
PREMISE OF THE STUDY: Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. METHODS: We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. KEY RESULTS: Concatenation-and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. CONCLUSIONS: Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow. KEY WORDS coalescence; chloroplast capture; hybrid; incomplete lineage sorting; low-copy nuclear loci; phylogenetic incongruence; Pinaceae; pine; plastome.
Discordance among gene trees across genomes is an important feature of organisms (Degnan and Rosenberg, 2009). Doyle (1992) and Maddison (1997) outlined how individual gene trees may disagree as a result of processes such as incomplete lineage sorting (ILS), reticulation, horizontal gene transfer, gene duplication, and paralogy. Of these, ILS has received the most attention and is predicted to be particularly relevant in species that have large effective population sizes, low mutation rates, long generation times, or that have undergone rapid radiations (Pamilo and Nei, 1988; Degnan and Rosenberg, 2009). Reticulation through hybridization or organellar capture has also been documented as playing an important role in gene-tree discordance (Rieseberg and Soltis, 1991). Pines are long lived and have large effective population sizes, making them an
ideal group for studying ILS (Syring et al., 2007; DeGiorgio et al., 2014). They are also wind pollinated, with weak barriers to interspecific gene flow, permitting hybridization to increase the proportion of alleles shared by reproductively compatible species (Mirov, 1967; Hong et al., 1993). Pinus subsection Australes Loudon comprises ~30 pine species of outstanding ecological and economic importance distributed in North and Central America and the Greater Antilles (Farjon, 2010; Fig. 1). The subsection can be subdivided into three groups (Table 1). The first, colloquially known as the California closed- cone pines, comprises three species with serotinous cones from western North America. Here, we refer to the group informally as the Attenuatae. Van der Burgh (1973) erected Pinus subsection
American Journal of Botany 105(4): 1–15, 2018; http://www.wileyonlinelibrary.com/journal/AJB © 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. • 1
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FIGURE 1. Geographic distribution of Pinus subsection Australes and collection localities for the material included in this study. Species distributions are from Critchfield and Little (1966) and digitized by the U.S. Geological Survey.
Attenuatae for these species, a rank that was adopted by Price et al. (1998) but not by Gernandt et al. (2005). The second group, the southern yellow pines, comprises ~11 species from the eastern United States and Antilles. We refer to the group as Australes sensu stricto (s.s.). The third group, known colloquially as the egg-coned pines, comprises ~16 species from Mexico and Central America. We refer to the group informally as the Oocarpae. Little and Critchfield (1969) erected Pinus subsection Oocarpae for most of its members. Economically important species of Pinus subsection Australes include loblolly pine (P. taeda), longleaf pine (P. palustris), Monterey pine (P. radiata), and Mexican weeping pine (P. patula). Pinus subsection Australes is a monophyletic group within Pinus section Trifoliae Duhamel (the North American hard pines), which also includes Pinus subsections Contortae Little and Critchfield and Ponderosae Loudon (Gernandt et al., 2005). Traditional classifications based on morphology, anatomy, and geography did not circumscribe the species in section Trifoliae, instead placing them among two or more infrageneric taxa that included more distantly related hard pines of North America or the Old World (Pinus subgenus Pinus; Shaw, 1914; Pilger, 1926; Little and Critchfield, 1969; Van der Burgh, 1973). Section Trifoliae can be recognized by a combination of nonexclusive morphological characters, notably needles with two vascular bundles, persistent fascicle sheaths in most species, seed cone scales with a dorsal umbo, and seeds with articulate wings (Price et al., 1998; Gernandt et al., 2005). No known morphological characters unequivocally distinguish subsection Australes
from its presumed sister group subsection Ponderosae, although in subsection Australes seed cones are more commonly ovate and serotiny is more widespread, and in the Oocarpae most species have cones on elongated peduncles. The fossil history of Pinus subsection Australes is poorly understood. According to Axelrod (1986), fossils with morphological similarities to these groups indicate that the subsection formerly was more widely distributed in North America, with an age extending to the Late Eocene–Early Oligocene. In the case of Australes s.s., Axelrod (1986) mentions a possible distribution in Eurasia. He also postulated that 30–25 mya, the Oocarpae extended into the present-day United States along mountain chains that included an “Appalachian-Ozarks-Trans-Pecos Texas” corridor. These taxa were hypothesized to have succumbed to climate-induced extinction, resulting in the present disjunction between taxa in Mexico and the southern United States. Molecular dating studies that incorporate Axelrod’s interpretations of the fossil record have estimated relatively early ages for the separation of subsection Australes from subsection Ponderosae (the stem age) and the earliest divergence within Australes (the crown age). For example, using a relaxed molecular clock with plastid DNA sequences, Eckert and Hall (2006) dated ten nodes within Pinus, including an interval constraint of 15–40 mya for the crown age of subsection Australes, obtaining a Paleocene age (63 mya) for the divergence of subsection Australes from subsection Ponderosae and an Eocene age (43 mya; beyond the lower limit
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TABLE 1. Taxa classified in Pinus subsection Australes and their geographic distribution. Taxon Attenuatae Pinus attenuata Lemmon P. muricata D. Don P. radiata D. Don var. radiata P. radiata var. binata (Engelm.) Lemmon Australes s.s. P. caribaea Morelet P. caribaea var. bahamensis (Griseb.) W.H. Barrett & Golfari P. caribaea var. hondurensis (Sénecl.) W.H. Barrett & Golfari P. cubensis Griseb. P. echinata Mill. P. elliottii Engelm. var. Elliottii P. elliottii var. densa Little & K.W. Dorman P. glabra Walter P. occidentalis Sw. P. palustris Mill. P. pungens Lamb. P. rigida Mill. P. serotina Michx. P. taeda L. Oocarpae P. chihuahuana Engelm. P. georginae Pérez de la Rosa P. greggii Engelm. ex Parl. var. Greggii P. greggii var. australis Donahue & Lopez P. herrerae Martínez P. jaliscana Pérez de la Rosa P. lawsonii Roezl ex Gordon P. leiophylla Schiede ex Schltdl. & Cham. P. lumholtzii B.L. Rob. & Fernald P. luzmariae Pérez de la Rosa P. oocarpa Schiede ex Schltdl. P. patula Schiede ex Schltdl. & Cham. var. patula P. patula var. longipedunculata Loock ex Martínez P. praetermissa Styles & McVaugh P. pringlei Shaw P. tecunumanii Eguiluz & J.P. Perry P. teocote Schiede ex Schltdl. & Cham. P. vallartensis Pérez de la Rosa & Gernandt
Distribution Western North America Western North America Western United States Baja California, Mexico
Western Cuba and Isla de la Juventud Bahamas Central America Cuba Southern United States Southern United States Southern United States Southern United States Caribbean Southern United States Southern United States Southern United States Southern United States Southern United States Mexico and southwestern United States Western Mexico Eastern Mexico Eastern Mexico Western Mexico Western Mexico Mexico Mexico Western Mexico Western Mexico Mexico and Central America Eastern and central Mexico Southern Mexico Western Mexico Mexico Mexico Mexico Western Mexico
of their constraint) for the crown age. More recently, Saladin et al. (2017) used node and tip dating of plastid DNA sequences based on 21 Pinus fossils and obtained a range of ages for the crown divergence of Pinus subsection Australes that were centered in the early Miocene (~20 mya). Molecular clock estimates based on nuclear DNA that used fewer fossil calibration nodes, none of which were within the North American hard pines, obtained much younger age estimates of 7 and 13 mya for the crown of subsection Australes (Willyard et al., 2007; 8 or 15 mya for the stem). Similarly, using two different calibration hypotheses that dated nodes outside the North American hard pines obtained a crown age estimate for subsection
Australes of 5 or 6 mya (and a stem age of 10 and 12 mya) on a plastid DNA tree (Gernandt et al., 2008). These younger age estimates corresponding to the second half of the Miocene imply a more rapid diversification of the ~30 species of the subsection and a greater potential role for ILS in shaping genetic relationships. The ages also are in correspondence with climate-induced vicariance events identified in other boreal taxa whose distributions include disjunct areas in Mexico and the southeastern United States (e.g., Morris et al., 2008). Phylogenetic studies of Pinus subsection Australes have found contradictory results using different markers. Adams and Jackson (1997) investigated the phylogenetic and biogeographic relationships of Australes s.s. (the Oocarpae were not included) based on 22 morphological characters. The tree topologies that they recovered were consistent with a migration from the southeastern United States to the Antilles. An analysis of partial sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA recovered six species of subsection Australes in a weakly supported clade with subsection Contortae (Liston et al., 1999, 2003). A neighbor- joining analysis of 127 RAPD markers in 17 species (following the classification in Table 1) of Pinus subsection Australes recovered one of the three principal lineages, the Attenuatae, as monophyletic, and the Australes s.s. and Oocarpae as incompletely resolved from one another (Dvorak et al., 2000). Plastid DNA studies have recovered subsection Australes as monophyletic with respect to subsections Contortae and Ponderosae but did not recover the Attenuatae, Australes s.s., or Oocarpae as monophyletic (Gernandt et al., 2005; Hernández-León et al., 2013). The study by Hernández-León et al. (2013) featured several striking results, including the grouping of P. glabra from the southeastern United States with the California closed-cone pines, and the grouping of P. chihuahuana and P. leiophylla with the morphologically dissimilar P. greggii. Furthermore, of 95 individuals representing 29 species included, plastid haplotypes formed monophyletic groups for only 16 of the species. ILS and introgression were hypothesized to explain the lack of monophyly of plastid sequences by species, but no attempt was made to disentangle the two causes. Pinus subsection Australes offers an opportunity to address the challenge of inferring species phylogenies in a group with slow coalescence rates and interspecific gene flow. We used targeted sequence capture (Gnirke et al., 2009) combined with genome skimming (Hyb-Seq; Weitemier et al., 2014) to characterize nuclear and plastid DNA sequences from multiple individuals per species and perform concatenated and multispecies coalescent analyses. Our aims were to infer phylogenetic relationships within and between the hypothesized principal lineages of the subsection (Attenuatae, Australes s.s., and Oocarpae) and to investigate the possible causes of nuclear and cytoplasmic incongruence. MATERIALS AND METHODS Study material
We sampled 72 individuals representing 27 of 30 recognized species from Pinus subsection Australes, including three infraspecific taxa (Table 1; Appendix S1; see Supplemental Data with this article). Pinus caribaea and the newly described P. vallartensis (Pérez de la Rosa and Gernandt, 2017) were not included, and P. taeda was represented only by sequences published by Neves et al.
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(2013). Twenty-six of the individuals were included in a previous plastid DNA study (Hernández-León et al., 2013). Pinus subsection Ponderosae was recovered as the sister group in previous phylogenetic analyses (e.g., Gernandt et al., 2005; Willyard et al., 2007). We included two species from subsection Ponderosae, Pinus devoniana and P. douglasiana, as outgroups. We extracted pine DNA using the CTAB method (Doyle and Doyle, 1987) for diploid leaf tissue or a Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin, USA) for haploid seed megagametophyte tissue (Appendix S2). A Qubit fluorometer and Qubit dsDNA HS assay kit (Life Technologies, Carlsbad, California, USA) were used to measure DNA concentration, and a Nanodrop spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) was used to measure the absorbance maxima ratio. Samples with ≥800 ng of DNA and an A260/A280 between 2.0 and 2.2 were carried on to the next step. Marker selection
A total of 1045 putative single-copy nuclear genes were screened for probe design. Six were selected from a previous phylogenetic study of Pinus subsection Ponderosae (Willyard et al., 2009), three from a conserved ortholog set for Pinaceae (Liewlaksaneeyanawin et al., 2009), and 1036 from an exonic study of Pinus taeda and P. elliottii (Neves et al., 2013). The latter gene sequences were chosen from 14,729 putative unigenes in P. taeda, from which 11,396 gene models could be constructed from de novo assembly after exon hybridization capture (Neves et al., 2013). Except for four of the six genes selected from Pinus subsection Ponderosae (4CL, LEA, PAL, and WD40), we only considered those with a length >850 bp. Gene ontology annotations for “binding,” “cell wall,” “leaf,” “membrane,” “resistance,” “stress,” “root development,” and “root hair elongation” were used as criteria for selecting 940 of these genes. The remaining 96 genes were chosen because they could be related to a linkage map study of P. taeda (Echt et al., 2011). The exon sequences were submitted to Arbor Biosciences (formerly Mycroarray, Ann Arbor, Michigan, USA) for the development of 120 bp candidate RNA baits for each gene (~20 per gene). The probe sequences were used to perform a BLAST search on the P. taeda draft genome (version 1.0; https://genomevolution.org/coge/). Baits were eliminated if they had ≥10 hits to the P. taeda genome at a Tm of 62.5–65°C and ≥4 hits above 65°C, and if they were not flanked by two consecutive baits on each side that had been selected. Genes with bait coverage along
