Progenitor–Derivative Speciation in Pozoa (Apiaceae, Azorelloideae) of the Southern Andes

Annals of Botany Page 1 of 13 doi:10.1093/aob/mcr291, available online at www.aob.oxfordjournals.org

Progenitor–derivative speciation in Pozoa (Apiaceae, Azorelloideae) of the southern Andes

Patricio Lo´pez1,*, Karin Tremetsberger2, Gudrun Kohl1 and Tod Stuessy1 1Department of Systematic and Evolutionary Botany, Faculty Center of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria and 2Institute of Botany, Department of Integrative Biology and Biodiversity Research, University of Natural Resources and Life Sciences, Gregor-Mendel-Straße 33, 1180 Vienna, Austria * For correspondence. E-mail [email protected]

Received: 18 June 2010 Returned for revision: 5 January 2011 Accepted: 20 October 2011 Downloaded from † Background and Aims Studies examining patterns and processes of speciation in South America are fewer than in North America and Europe. One of the least well documented processes has been progenitor–derivative speciation. A particularly instructive example occurs in the southern Andes in the genus Pozoa (Apiaceae, Azorelloideae), which consists of only two diploid outcrossing species, the widespread P. coriacea and the geo- graphically and ecologically restricted P. volcanica. This paper tests the hypothesis that the latter species originated from the former through local geographical and ecological isolation by progenitor–derivative speciation. http://aob.oxfordjournals.org/ † Methods DNA sequences were analysed from Pozoa and the related South American genera Asteriscium, Eremocharis and Gymnophyton from non-coding regions of the plastid genome, ndhF-rpl32 and rpl32-trnL, plus incorporation of previously reported rpl16 intron and trnD-trnT intergenic spacer sequences. Ampliﬁed fragment length polymorphism (AFLP) data from 105 individuals in 21 populations throughout the entire range of distribution of the genus were used for estimation of genetic diversity, divergence and SplitsTree network analysis. Ecological factors, including habitat and associated species, were also examined. † Key Results Pozoa coriacea is more similar genetically to the outgroup genera, Asteriscium and Eremocharis, than is P. volcanica. At the population level, only P. volcanica is monophyletic, whereas P. coriacea is paraphy-

letic. Analyses of genetic differentiation among populations and genetic divergence and diversity of the species at World Trade Institute on September 26, 2014 show highest values in P. coriacea and clear reductions in P. volcanica. Pozoa coriacea occurs in several types of high elevation habitats, whereas P. volcanica is found only in newly formed open volcanic ash zones. † Conclusions All facts support that Pozoa represents a good example of progenitor–derivative speciation in the Andes of southern South America.

Key words: AFLP, Andes mountains, Apiaceae, DNA sequencing, genetic diversity, geographical origin, Pozoa coriacea, P. volcanica, speciation.

INTRODUCTION This concept includes vicariance and peripatric speciation. In the former, environmental change produces a barrier that Speciation relates to formation of spatial or geographical bar- divides the original geographical range of a species into two riers and/or ecological and reproductive isolating mechanisms or more populations, reproductive isolation developing in that allow emergence of new gene combinations in different each of them. In peripatric speciation, reproductive isolation populations, generating differences between populations that originates by colonization of an unoccupied habitat by a few eventually produce new species (Grant, 1981; Coyne, 1992; individuals (Coyne and Orr, 2004; Lomolino et al., 2006). In Gavrilets, 2003; Levin, 2003). The use of information from this case, new populations become spatially separated from geographical, ecological, reproductive and chromosomal the progenitor, and divergence ensues (Tauber and Tauber, studies has allowed a considerable theoretical framework to 1989). Sympatric speciation involves evolution of reproductive be developed over the past century to explain speciation in isolation within the average dispersal distance of a single indi- plants and animals. In the past two decades, the advent of vidual (Mayr, 1963; Bolnick and Fitzpatrick, 2007), and where molecular methods [sequencing of DNA, amplified fragment the restriction of gene flow originates through biological char- length polymorphism (AFLP) analyses, microsatellites and acteristics of the organisms (Futuyma and Mayer, 1980). single nucleotide polymorphisms] has allowed a much A particular type of peripatric speciation, whereby an iso- broader understanding of the genetic patterns involved with lated peripheral population diverges to form a derivative speciation (Coyne and Orr, 2004). species, has been called progenitor–derivative speciation Different modes of geographical speciation are known to (Crawford and Smith, 1982). The derivative species diverges have occurred among higher plants. Allopatric speciation from the ancestral condition, but the progenitor species takes place when a geographical boundary reduces gene flow remains almost unchanged. This differs from typical geograph- between ancestral populations and leads to the formation of ic allopatric speciation, whereby two populations diverge reproductive barriers (Grant, 1981; Lomolino et al., 2006). simultaneously in numerous characters and the ancestor

# The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa disappears in the process (Gottlieb, 1973; Jaramillo-Correa species (Mathias and Constance, 1962). Pozoa coriacea is and Bousquet, 2003). Progenitor–derivative speciation may widespread at elevations between 1000 and 4000 m and dis- be more recent in comparison with gradual divergence under tributed along the southern Andes in Chile from Coquimbo the classical allopatric model. Thus, it should be somewhat south to the Region de La Araucanıá and from the Province easier to identify differences between the taxa associated of San Juan south to Rio Negro on the Argentinean side. with progenitor–derivative speciation as compared with Pozoa volcanica is restricted in distribution, growing those undergoing gradual divergence after speciation, given between 1200 and 2400 m only in the Lonquimay region that the progenitor species presumably has changed little and surrounding area in southern Chile, plus the adjacent since the derivative species originated (Crawford, 2010). Province of Mendoza and Neuqueń in Argentina (Mathias Criteria that have been suggested for identifying cases of and Constance, 1962; Martıńez, 2008). The restricted geo- progenitor–derivative speciation include the following (after graphical distribution of P. volcanica near the centre of the Crawford, 2010): (1) a close morphological and phylogenetic range of P. coriacea, the similar morphology of the two relationship should exist between the two taxa (i.e. they species and the existence of only two species within this mor- should be sister species); (2) crossability barriers between phologically distinct genus suggest the hypothesis that

the population systems and/or observed karyotypic differences P. volcanica arose through a process of progenitor–derivative Downloaded from may prevail; (3) lower genetic diversity should occur in the speciation from P. coriacea. derivative species in comparison with the progenitor; (4) the This paper tests the hypothesis of progenitor–derivative derivative populations should nest phylogenetically within speciation within Pozoa with the following specific objectives: those of the progenitor taxon; and (5) characteristics of the (1) confirming that Pozoa is a monophyletic genus; (2) deter- derivative populations may suggest directional evolution, mining if one species of Pozoa is ancestral to the other and, if such as reduction in allelic richness, breakdown of self- so, which; and (3) investigating levels of genetic divergence http://aob.oxfordjournals.org/ incompatibility, occurrence on ultramafic soils or restricted and variation in the derived species in comparison with its pro- distribution at the periphery of the more broadly ranging progenitor. To complete these objectives, we have selected DNA genitor species. sequencing from the plastid genome and AFLP analysis (Vos Only a few cases of progenitor–derivative speciation in et al., 1995). The latter is particularly efficacious for revealing plants have so far been documented, principally all in the patterns of genetic variation in natural populations (Gaudeul Northern Hemisphere: Stephanomeria malheurensis from et al., 2000; Nybom, 2004; Andrade et al., 2009) and for Stephanomeria exigua subsp. coronaria (Asteraceae; for a revealing genetic structure of intra- and interspecific taxa

review, see Gottlieb, 2003); Clarkia lingulata from C. biloba (Wooten and Tolley-Jordan, 2009). These sensitive AFLP at World Trade Institute on September 26, 2014 (Onagraceae; Gottlieb, 1974); Coreopsis nuecensis from its markers, to our knowledge, have not yet been applied to exam- progenitor species C. nuecensoides (Asteraceae; Crawford ination of progenitor–derivative speciation. and Smith, 1982); Lasthenia maritima from its progenitor L. minor (Asteraceae; Crawford et al., 1985); Layia discoidea originating from L. glandulosa (Asteraceae; Gottlieb et al., MATERIALS AND METHODS 1985; Baldwin, 2005); Camassia angustata from The species C. scilloides (Asparagaceae; Ranker and Schnabel, 1986) and Picea rubens from P. mariana (Pinaceae; Pozoa coriacea Lag. (Fig. 1A; common name ‘Anislao’ or Jaramillo-Correa and Bousquet, 2003). ‘Asta de cabra’) is an outcrossing perennial herb with a In South America, numerous tectonic changes and elevation large, underground stem divided into many slender lateral divi- of the Andes mountain chain have stimulated allopatric speci- sions, and with spreading-ascending to recurved terminal flow- ation in different plant groups. However, only a handful of ering stalks (peduncles). Leaves are ovate to orbicular– papers have examined speciation by means of species-level reniform or obovate, undulate or slightly to roughly dentate, phylogenetic analyses using molecular methods such as in usually with 3–15 shallow teeth. Umbels have 20–35 Perezia (Asteraceae; Simpson, 1973; Simpson et al., 2009), flowers, some staminate. Flowers are usually purplish or Nothofagus (Nothofagaceae; Manos, 1997), Fragaria purple. Fruits are oblong–ovate to cuneate–oblong, with the (Rosaceae; Ontivero et al., 2000), Malesherbia (Passifloraceae; mature carpels slightly compressed (Matthias and Constance, Gengler-Nowak, 2002, 2003), Hypochaeris (Asteraceae, 1962). The chromosome number is 2n ¼ 20 (Bell and Samuel et al. , 2003; Stuessy et al.,2003; Tremetsberger et al., Constance, 1957; Rahn, 1960). 2005, 2006), Chaetanthera (Asteraceae, Hershkovitz et al., Pozoa volcanica Mathias & Constance (Fig. 1B) is also an 2006), Tristerix (Loranthaceae, Amico et al.,2007), Drimys outcrossing perennial herb, with a large and undivided under- (Winteraceae; Ruiz et al., 2008)andPuya (Bromeliaceae; ground stem, but it has a short and enlarged terminal peduncle. Schmidt and Systma, 2010). These studies have suggested Leaves are ovate–orbicular to reniform, slightly or doubly avenues for synthesis of molecular, ecological, reproductive dentate, usually with 13–30 triangular teeth. Umbels have and biogeographical aspects, all of which are beginning to 25–45 flowers, some staminate. Flowers are usually provide new understanding of evolutionary processes in the greenish-yellow. The fruits are oblong–ovate, the mature Andes of South America. carpels being strongly compressed. The chromosome number An example of possible progenitor–derivative speciation in is also 2n ¼ 20 (Bell and Constance, 1957; Rahn, 1960). South America occurs in the genus Pozoa (Apiaceae, The principal morphological differences between the two Azorelloideae), endemic to the Andes of Chile and species are the stem with slender divisions in P. coriacea, Argentina (Figs 1 and 2). This genus consists of only two the greater number of teeth on the leaves of P. volcanica and Lo´pez et al. — Progenitor–derivative speciation in Pozoa Page 3 of 13

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F IG. 1. Species of the genus Pozoa and their typical habitats in southern South America. (A) Pozoa coriacea; (B) P. volcanica; (C) Chile, Region del Maule, Laguna Teno; (D) Chile, Region de la Araucanıá, Navidad cone (centre), Volcań Lonquimay. Scale bars ¼ 3 cm. the flowers being usually greenish-yellow in P. volcanica and Sequences purplish in P. coriacea. at World Trade Institute on September 26, 2014 Genomic DNA was extracted from individuals in 27 populations belonging to seven species in the genera Asteriscium, Eremocharis, Gymnophyton and Pozoa (Table 1) from silica Sampling gel-dried leaf material following the cetyltrimethylammonium Twenty-two populations of Pozoa were collected throughout bromide (CTAB) method (Doyle and Doyle, 1987) with minor the entire range of the two species (Fig. 2, Table 1), extending modifications (Tremetsberger et al., 2003). We tested several from Portillo in the north of Chile to La Hoya in the south of plastid sequences (3′rps16-5′trnK, trnH-psbA, matK, Argentina for P. coriacea (11 populations), and within the ndhF-rpl32 and rpl32-trnL; Shaw et al., 2007) for their poly- Lonquimay region in southern Chile and adjacent Mamuil morphism on two or three individuals of P. coriacea and Malal in Argentina for P. volcanica (11 populations). Leaves P. volcanica. This primer trial suggested the two non-coding of five individuals from each population were collected in plastid regions, ndhF-rpl32 and rpl32-trnL, corresponding to silica gel. Vouchers of each population sampled are on the intergenic spacer and located in the small single-copy deposit in the herbarium of the University of Vienna (WU). region as most informative. Amplifications were made using The populations of P. coriacea grow in different substrates the following primers: ndhF (5′-GAA AGG TAT KAT CCA (Fig. 1C), such as stable volcanic soil, black or clay soil, red YGM ATA TT-3′)/rpl32-R (5′-CCA ATA TCC CTT YYT gravel, sand and between rocks. Genera of the high Andean TTT CCA A-3′), and rpl32-F (5′-CAG TTC CAA AAA vegetation that accompany P. coriacea include: Mulinum AAC GTA CTT C-3′)/trnL(UAG) (5′-CTG CTT CCT AAG (Apiaceae); Araucaria (Araucariaceae); Baccharis, Chuquiraga, AGC AGC GT-3′), respectively (Shaw et al., 2007). Hypochaeris, Mutisia and Nassauvia (all Asteraceae); Berberis PCRs were carried out using 0.4mM of each primer and (Berberidaceae); Empetrum (Ericaceae); Adesmia and Lathyrus ReddyMix PCR Master Mix (ABgene, Vienna, Austria) in- (Fabaceae); Nothofagus (Fagaceae); Polygonum (Polygonaceae); cluding 2.5mM MgCl2 (according to the manufacturer’s Acaena (Rosaceae); Nertera (Rubiaceae); Calceolaria instructions). Amplifications were performed in a GeneAmp (Calceolariaceae); and Tropaeolum (Tropaeolaceae). PCR System 9700 (Applied Biosystems) with an initial Pozoa volcanica grows in new volcanic ash (Fig. 1D), with 5 min at 80 8C followed by 36 cycles each of 30 s denaturation porous rock and pebbles, and occasionally black or brown soil. at 95 8C, 30 s annealing at 50 8C, an elongation phase of 4 min Accompanying vegetation includes the genera: Baccharis, at 65 8C, followed by a final elongation phase of 5 min at Hypochaeris, Nassauvia and Senecio (Asteraceae); Adesmia 65 8C. PCR products were purified using 0.5 mL of exonucle- and Trifolium (Fabaceae); Loasa (Loasaceae); Chusquea ase I from Escherichia coli and 1 mL of shrimp alkaline phos- (Poaceae); Polygonum and Rumex (Polygonaceae); and phatase (Fermentas) for 45 min at 37 8C followed by enzyme Acaena (Rosaceae). inactivation for 15 min at 85 8C. Cycle sequencing was Page 4 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa

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10 17 15 19 16 18 20

40° S

CHILE 22 01428 21 km

ARGENTINA

035 70 140 km

70° W

F IG. 2. Distribution of sampled populations of Pozoa coriacea (squares) and P. volcanica (circles) in southern South America. Generalized distributions of P. coriacea and P. volcanica are shown by dashed red and blue lines, respectively. Lo´pez et al. — Progenitor–derivative speciation in Pozoa Page 5 of 13

TABLE 1. Collection data for populations of Pozoa and generic relatives for sequencing (S) and AFLP (A) studies

Collection Elevation EMBL accession Species Analysis Population number Latitude Longitude (m) number

Pozoa coriacea Lag. A, S 1: Chile, Portillo PL et al. 2605 32 850′09′′S70807′43′′W 2880 FR871934 A 2: Chile, Valle PL et al. 2531 33 820′01′′S70814′49′′W 3100 – Nevado A 3: Chile, Embalse El PL et al. 2601 33 837′05′′S69858′04′′W 2580 – Yeso A 4: Chile, Laguna Teno PL et al. 2600 35 809′48′′S70832′17′′W 2540 – A 5: Chile, Banõs de PL et al. 2604 35 850′01′′S69859′25′′W 2438 – Colina A 6: Chile, Laguna del PL et al. 2593 36 800′54′′S70830′20′′W 2360 – Maule A, S 7: Chile, Chillań, PL et al. 2548 36 852′34′′S71827′51′′W 1550 FR871933, FR871941 Shangri-La ′ ′′ ′ ′′ A 8: Argentina, Volcań KT et al. 1033 37 849 53 S71806 44 W 2120 – Downloaded from Copahue A 9: Chile, Volcań KT et al. 1026 37 854′42′′S71824′00′′W 1675 – Callaquı´ A 10: Chile, Termas de PL et al. 2564 38 834′34′′S71837′38′′W 1035 – Rio Blanco A, S 11: Argentina, La PL et al. 2686 42 849′58′′S71815′26′′W 1660 FR871940 Hoya http://aob.oxfordjournals.org/ Pozoa volcanica Math. & Const. A 12: Chile, Cerros de KT et al. 135 38 820′55′′S71825′52′′W 1820 – Lanco A 13: Chile, Cerro KT et al. 16 38 824′40′′S71834′34′′W 1880 – Colorado A 14: Chile, Cordillera KT et al. 138826′29′′S71828′48′′W 1800 – Las Raıćes A 15: Chile, Sierra KT et al. 61 38 837′02′′S71835′50′′W 1730 – Nevada A 16: Chile, Pino PL et al. 2559 38 839′20′′S70855′11′′W 1770 –

Hachado B at World Trade Institute on September 26, 2014 A 17: Chile, Pino KT et al. 130 38 839′40′′S70853′53′′W 1900 – Hachado A A, S 18: Argentina, Pino PL et al. 2677 38 840′03′′S70850′46′′W 1558 FR871943 Hachado C A, S 19: Chile, Conguillıó PL et al. 2565 38 840′59′′S71848′01′′W 1440 FR871942 A 20: Chile, Volcań KT et al. 106 38 841′26′′S71847′06′′W 1725 – Llaima A, S 21: Argentina, PL et al. 2680 39 836′26′′S71821′50′′W 980 FR871936 Mamuil Malal S 22: Chile, Volcań PL et al. 2578 39 835′47′′S71830′20′′W 1380 FR871935 Lanin Asteriscium chilense Cham. & S 23: Chile, Puerto PL et al. 2517 31 821′39′′S71835′21′′W 230 FR871928, FR871938 Schlecht. Oscuro Asteriscium vidali Phil. A, S 25: Chile, Huasco, PL et al. 2508 28 829′28′′S71814′07′′W 340 FR871929, FR871939 Cerro Negro Eremocharis fruticosa Phil. S 26: Chile, Quebrada PL et al. 2505 25 801′50′′S70826′15′′W 710 FR871930, FR871937 Peralito Gymnophyton foliosum Phil. S 27: Chile, Taltal, PL et al. 2504 25 823′05′′S70826′42′′W 40 FR871931, FR871944 Quebrada San Ramoń Gymnophyton isatidicarpum A, S 28: Chile, Vicunã PL et al. 2509 30 808′42′′S70830′17′′W 1300 FR871932 (Presl ex DC.) Math. & Const.

Vouchers are on deposit at the University of Vienna. PL, Patricio Lo´pez; KT, Karin Tremetsberger. performed for the forward and reverse strand with 0.7 mLof manual adjustments using the program BioEdit version 7.0.9.0 BigDye Terminator v3.1 Ready Reaction Mix (Applied (Hall, 1999). Indels were treated as binary characters following Biosystems) in a 10 mL total volume ﬁlled up with water, the ‘simple indel coding method’ (Simmons and Ochoterena, 1 mL of forward or reverse primer and 6.8 mL of PCR 2000) using the program SeqState version 1.36 (Mu¨ller, product with the following conditions: 1 min at 96 8C followed 2005). A heuristic search for most parsimonious (MP) trees by 35 cycles of 10 s at 96 8C, 5 s at 50 8C and 4 min at 60 8C. was performed with PAUP* version 4.0b8 (Swofford, 2002). Sequencing reactions were analysed on a capillary sequencer The analyses involved 1000 replicates with stepwise random (3730 DNA Analyzer; Applied Biosystems). taxon addition, tree bisection–reconnection (TBR) and branch The sequences were assembled and aligned using Seqman II swapping saving no more than ten trees per replicate. All (DNASTAR) and Clustal (Thompson et al., 1997), followed by characters were equally weighted and treated as unordered Page 6 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa

(Fitch, 1971). Clade support was estimated using non- Shannon Diversity Index HSh ¼ –S[ pi × ln( pi)], where pi is parametric bootstrapping (Felsenstein, 1985)with10000 the frequency of the ith band in the respective population bootstrap replicates each with ten random sequence addition based on all AFLP bands recorded using FAMD ver. 1.108 replicates holding maximally ten trees per replicate, TBR (Schlu¨ter and Harris, 2006). The Pearson correlation was branch swapping and MulTrees on. A phylogenetic supertree used to test correlation among different genetic diversity was built from the ndhF-rpl32 and rpl32-trnL trees using the estimates using SPSS ver. 15.0(#SPSS Inc.). The matrix representation with parsimony method implemented in Mann–Whitney U-test was used to estimate the signiﬁcance the program SuperTree 0.85b (Salamin et al.,2002)withthe of differences of genetic divergence and diversity of popula- following options: coding scheme, Baum/Ragan; and character tions between species using SPSS. type, unordered. EMBL accession numbers of the DNA A Nei–Li distance matrix was calculated from the AFLP sequences are presented in Table 1. matrix, and used as input for the phylogenetic network with the NeighborNet algorithm (Bryant and Moulton, 2004), as implemented in the software SplitsTree ver. 4.10 (Hudson AFLP ﬁngerprinting and Bryant, 2006). A Neighbor–Joining tree based on the

We scored 105 individuals of Pozoa and two individuals of Nei–Li distance matrix was condensed and midpoint-rooted Downloaded from the outgroup (Asteriscium and Gymnophyton) for three AFLP using FigTree ver. 1.3.1(Rambaut, 2006–2009). primer combinations. Genomic DNA was extracted from silica gel-dried leaf material following the CTAB method (Doyle and Doyle, 1987) with minor modifications (Tremetsberger Estimation of genetic differentiation et al., 2003). The AFLP protocol followed Vos et al. (1995) Genetic differentiation among species was evaluated by ana- with modifications as indicated in Tremetsberger et al. lysis of molecular variance (AMOVA) using Arlequin ver. 3.1 http://aob.oxfordjournals.org/ (2003). The selective primer combinations chosen following (Excoffier et al., 2006); total genetic diversity was partitioned primer trials are MseI-CTGA/EcoRI-ACT (Fam), MseI-CTT/ into components among two hierarchical levels, among popu- EcoRI-ACT (Vic) and MseI-CAC/EcoRI-ACC (Ned). lations (FST) and among individuals within populations. An The presence and absence of bands in all individuals were alternative Bayesian approach (Holsinger et al., 2002)was scored with GeneMarker ver. 1.85 by Soft Genetics. For raw used to obtain an independent estimate of FST for each popu- data analysis of each primer combination, local southern size lation. This method allows estimation of FST from dominant call algorithm, smooth peak saturation, baseline subtraction, markers without assuming Hardy–Weinberg proportions in pull-up correction and spike removal were selected. We used populations. The original data matrix was imported into at World Trade Institute on September 26, 2014 the range 150–510 bp for all primer combinations. The peak Hickory ver. 1.1(Holsinger and Lewis, 2003–2007) and detection threshold was an intensity of relative fluorescent used for a full model, f ¼ 0 model, theta ¼ 0 model and the units .50, with the percentage of relative minimum intensity f-free model run with default parameters (i.e. the hickory of allele peaks at 5 and with the same value for local region block omitted). The f-free model, which estimates theta percentage. The maximum relative fluorescent units threshold without estimating f (thus incorporating all the uncertainty in of peak height for peak detection was 30 000. Size calibration the prior of f ), is available for dominant marker data, was manually adjusted in some samples with values below because estimates of f derived from dominant marker data 90 %. The electropherograms were standardized using the may be unreliable. The deviance information criterion (DIC; automatic panel editor, generating a new panel for each Spiegelhalter et al., 2002) was used to estimate how well a par- colour. Each primer combination generates a binary matrix, ticular model fits the data and to choose between models. combined in one for analysis of genetic diversity and differentiation (Wooten and Tolley-Jordan, 2009). RESULTS Estimation of genetic diversity Sequence relationships The number of different AFLP phenotypes present in a The aligned ndhF-rpl32 region is 1231 bp long. Phylogenetic population was counted with Arlequin ver. 3.1(Excoffier analysis shows that 23 characters are parsimony uninformative et al., 2006). The number of private bands in each population and 27 are potentially parsimony informative. The alignment and species was calculated using FAMD ver. 1.108 (Schlu¨ter resulted in 18 potentially informative indels, one of which and Harris, 2006), and the Rarity Index, calculated by using was nested. The single most parsimonious tree [length 52, con- the R-script AFLPdat (Ehrich, 2006). For each individual, sistency index (CI) 0.962, rescaled consistency index (RC) each AFLP marker is divided by the total number of occur- 0.928] for the region ndhF-rpl32 among Asteriscium, rences of this marker in the data set. These relative values Eremocharis, Gymnophyton and Pozoa (Fig. 3A) shows that are then summed to the Rarity Index for this particular individ- Pozoa is most closely related to Gymnophyton [bootstrap ual. Population values are estimated as the average of the indi- (BS) ¼ 100 %] and that P. volcanica appears to be derived vidual values, and species values are estimated as the average from P. coriacea, but with low support. Pozoa volcanica of the population values. forms a clade, but P. coriacea is paraphyletic. The aligned Genetic diversity was assessed for each population and rpl32-trnL region is 894 bp long. Phylogenetic analysis species by using the total number of AFLP bands, percentage shows that 13 characters are parsimony uninformative and 30 of polymorphic bands (by dividing the number of polymorphic are potentially parsimony informative. The alignment resulted bands by the total number of bands in the data set) and in 13 potentially informative indels. The single most Lo´pez et al. — Progenitor–derivative speciation in Pozoa Page 7 of 13

A Pozoa volcanica 2565 B Pozoa volcanica 2578 91 75

Pozoa volcanica 2680 75 Pozoa volcanica 2677 100

Pozoa coriacea 2548 100 Pozoa coriacea 2686 64 99 Pozoa coriacea 2605 100 Pozoa coriacea 2548 Asteriscium chilense 2517 100 100 97 Gymnophyton foliosum 2504 Asteriscium vidali 2508

Asteriscium vidali 2508 Gymnophyton foliosum 2504 Downloaded from

Asteriscium chilense 2517 Gymnophyton isatidicarpum 2509

Eremocharis fruticosa 2505 Eremocharis fruticosa 2505

Asteriscium closii http://aob.oxfordjournals.org/ C D Gymnophyton isatidicarpum Pozoa volcanica Gymnophyton polycephalum Gymnophyton spinosissimum Pozoa volcanica Gymnophyton robustum Pozoa coriacea 2686 100 Asteriscium chilense Asteriscium glaucum Pozoa coriacea 2548 >95 Pozoa volcanica at World Trade Institute on September 26, 2014 73 Pozoa coriacea Pozoa coriacea 2605 Oschatzia cuneifolia Oschatzia saxifraga Gymnophyton foliosum 2504 Eremocharis longiramea Eremocharis tripartita Asteriscium vidali 2508 Eremocharis triradiata 100 Domeykoa amplexicaulis Asteriscium chilense 2517 Eremocharis fruticosa Domeykoa oppositifolia Eremocharis fruticosa 2505

F IG. 3. Phylogenetic relationships among the genera Asteriscium, Eremocharis, Gymnophyton and Pozoa based on plastid DNA markers (values above branches represent bootstrap values): (A) ndhF-rpl32, 50 % majority rule consensus tree of 10 000 bootstrap replicates based on maximum parsimony; (B) rpl32-trnL, 50 % majority rule consensus tree of 10 000 bootstrap replicates based on maximum parsimony; (C) rpl16 combined with trnD-trnT, redrawn from Nicolas and Plunkett (2009) (maximum likelihood and Bayesian inference); and (D) supertree using ndhF-rpl32 and rpl32-trnL trees based on maximum parsimony. Numbers after species names in A and B are collection numbers (see Table 1). parsimonious tree (length 44, CI 0.977, RC 0.958; Fig. 3B) 406, of which 405 (99.7 %) are polymorphic. Pozoa coriacea reveals the two species of Pozoa in one clade (BS ¼ 100 %) presents a total of 355 bands, of which 354 are polymorphic, connecting with the clade of Asteriscium (BS ¼ 99 %). The whereas P. volcanica has a total of 253 bands, with 246 being results of Nicolas and Plunkett (2009), using the plastid polymorphic. The number of fragments for all individuals and rpl16 intron and trnD-trnT intergenic spacer (Fig. 3C), by species (P. coriacea/P. volcanica)are142(130/84)for revealed Pozoa as monophyletic, and related to Asteriscium primer MseI-CTGA/EcoRI-ACT, 165 (147/97) for MseI-CTT/ and Gymnophyton. The four plastid sequences taken together, EcoRI-ACT and 99 (78/72) for MseI-CAC/EcoRI-ACC. All therefore, show Pozoa as a monophyletic genus, related to individuals had unique AFLP phenotypes. Gymnophyton and Asteriscium (Fig. 3C, D). Genetic diversity and divergence of populations. The total number of AFLP bands is slightly higher in P. coriacea than AFLP relationships in P. volcanica, but the percentage of polymorphic bands is Fragment patterns. The total number of AFLP bands found in higher in P. volcanica than in P. coriacea. The Shannon all individuals and all populations of both species of Pozoa is Diversity Index shows that both species have essentially the Page 8 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa same levels of within-population genetic diversity. The differ- DISCUSSION ences in the three estimates of diversity are not significant Monophyly of Pozoa according to the Mann–Whitney U-test (Table 2, Fig. 4A). A significant correlation was observed between these indices Before addressing the specific question of progenitor–derivative in both species; the Pearson correlation between the Shannon speciation in Pozoa, it is necessary to confirm that the genus is Diversity Index and the total number of bands is r ¼ 0.899 monophyletic. Although the morphology of Pozoa is unified [n ¼ 21, significance (two-tailed) ¼ 0.000] and between the and distinct (Fig. 1A, B), it is important to reject any consider- percentage of polymorphic bands and the total number of ation of biphyly involving related genera. bands it is r ¼ 0.675 [n ¼ 21, significance (two-tailed) ¼ Previous morphological and anatomical studies have sug- 0.001]. The correlation between the percentage of polymorph- gested which genera of Apiaceae might be the closest relatives ic bands and the Shannon Diversity Index is r ¼ 0.620 [n ¼ of Pozoa. Henwood and Hart (2001) completed a cladistic ana- 21, significance (two-tailed) ¼ 0.003]. Values of the mean lysis using morphological and anatomical data with a focus on for Shannon Diversity are similar in both species. Regarding Australian Hydrocotyloideae, but also including genera from estimates of genetic divergence, the number of private bands other continents. In this study, Pozoa was generically distinct

and the Rarity Index are both significantly higher in popula- in possessing fused carpophores (free in the other genera), but Downloaded from tions of P. coriacea than in P. volcanica (Mann–Whitney it grouped most closely with Asteriscium due to shared non- U-test, Table 2, Fig. 4B). Both indices are positively corre- inflexed petal apices. This sub-group joined next to lated, with Pearson correlation r ¼ 0.809 [n ¼ 21, significance Eremocharis, Domeykoa and Gymnophyton, constituting the (two-tailed) ¼ 0.000]. Comparing values of estimates of ‘Pozoa clade’. Liu (2004), using 16 morphological and anatom- divergence and diversity in each species, P. coriacea has con- ical characters in cladistic analyses, obtained a consensus tree siderably higher values than P. volcanica for all measures that showed Pozoa generically distinct by a concave dorsal http://aob.oxfordjournals.org/ (Table 3). fruit surface but nearest to Asteriscium and Gymnophyton. Previous molecular studies have also suggested a relationship Genetic diversity between species. Analysis of molecular vari- of Pozoa with Asteriscium and Gymnophyton of southern South ance attributes 20.20 % variance (d.f. ¼ 1) between species America. Nicolas and Plunkett (2009) examined affinities and 79.80 % variance (d.f. ¼ 103) within populations of each among 40 genera of subfamily Hydrocotyloideae using plastid species. The same analysis, but for each species, shows sequences of the rpl16 intron and trnD-trnT intergenic spacer 54.48 % variance among populations in P. coriacea (d.f. ¼ regions. In this analysis with a combined data set (Fig. 3C), . ¼ 10) and 45 52 % variance (d.f. 44) within populations Pozoa appears sister to Asteriscium and Gymnophyton (in at World Trade Institute on September 26, 2014 [95 % confidence interval (CI) ¼ 51.4–57.4%].Pozoa volca- their paper labelled as the Gymnophyton sub-clade) with 100 nica presents 25.55 % variance among populations (d.f. ¼ 9) % bootstrap support and a posterior probability of 1.0. and 74.45 % (d.f. ¼ 40) variance within populations (95 % In view of the importance of confirming monophyly in CI ¼ 22.1–28.9%). Pozoa, and following the suggestions of affinities revealed The genetic variance among species using a Bayesian ana- from previous studies, our own sequencing efforts focused ini- lysis shows the lowest DIC value with the f ¼ 0 model (DIC tially on examining relationships among Pozoa, Asteriscium, value ¼ 3242), where the theta-II value is 0.240 (95 % Domeykoa, Eremocharis and Gymnophyton. Primer trials CI ¼ 0.213–0.273). Among populations of P. coriacea (n ¼ recommended employment of the plastid markers 11; pop. 1–11), using the full model (DIC value ¼ 4576), ndhF-rpl32 and rpl32-trnL. Results of the former (Fig. 3A) the value of theta-II is 0.475 (95 % CI ¼ 0.453–0.498); in showed the closest relative to be Gymnophyton (100 % BS), P. volcanica (n ¼ 10; pop. 12–21; DIC value ¼ 3714) the and of the latter (Fig. 3B) to be Asteriscium (99 %). The theta-II value is 0.217 (95 % CI ¼ 0.191–0.243). studies of Nicolas and Plunkett (2009; Fig. 3C), using the NeighborNet analysis with SplitsTree using the whole rpl16 intron and trnD-trnT intergenic spacer, also showed a AFLP data set shows that the sister lineage (Asteriscium/ strong tie of Pozoa (100 % BS) to the genera Asteriscium Gymnophyton) links to Pozoa between P. coriacea populations and Gymnophyton. These two genera, therefore, were selected 1–10 and P. coriacea population 11 plus P. volcanica. When as outgroups for more detailed AFLP population-level ana- the SplitsTree is interpreted phylogenetically, this implies that lyses. All molecular data also point to Pozoa being monophy- population 11 is more closely related to P. volcanica than to letic. The previous studies (Fig. 3C) of Nicolas and Plunkett the remainder of P. coriacea (Fig. 5A, B). Of the two (2009) placed P. coriacea and P. volcanica together (.95 % species of Pozoa, the outgroup genera attach most closely to BS), as do our own results (Fig. 3A, B). AFLP analyses P. coriacea (Fig. 5B). Within P. coriacea there are two (Fig. 5) further support monophyly of Pozoa. NeighborNet groups that correspond to a general geographical trend. The analysis using SplitsTree of the many populations of both first group includes populations 1–7 of the central–north Pozoa spp., and including representatives of Asteriscium and part of the range, and the second covers populations 8–11 Gymnophyton, show substantial degrees of divergence of distributed in the southern zone. Population 11 of these genera in attachment to populations of P. coriacea. All P. coriacea (La Hoya) appears to be closely related to popula- data, therefore, support Pozoa as being monophyletic. tions 18 and 19 of P. volcanica, which may suggest an original geographical origin of P. volcanica from populations in this Ancestry of the species of Pozoa southern region of P. coriacea. The populations of P. volcanica do not show a clear geographical pattern In the context of monophyly of Pozoa, the next consider- (Fig. 5B). ation is the specific ancestry of the two included species. TABLE 2. Estimates of divergence and diversity based on AFLP analysis from five individuals in each of 21 populations of Pozoa coriacea and P. volcanica

Estimates of divergence Estimates of diversity Lo ´

Species Population No. of private bands Rarity Index Total no. of bands Percentage of polymorphic bands Shannon Diversity Index pez

Pozoa coriacea 154.70 93 14.53 17.13 — al. et 255.16 120 19.21 23.42 335.51 128 20.44 25.53 435.01 128 18.23 24.86

555.47 120 20.93 22.56 in speciation Progenitor–derivative 6137.63 152 26.35 33.32 744.89 135 22.17 26.11 842.89 81 14.78 18.69 942.25 68 12.56 15.69 10 1 2.16 78 12.56 14.58 11 19 7.46 129 14.78 29.99 Mean (+ s.d.) 6.00 (+ 5.25) 4.83 (+ 1.83) 112 (+ 27.33) 17.87 (+ 4.41) 22.90 (+ 5.94) Pozoa volcanica 12 2 2.66 100 24.13 23.13 13 4 2.96 100 18.23 22.82 14 1 2.40 95 17.49 20.69 15 1 1.95 83 16.50 17.92 16 1 3.32 122 21.18 28.55 17 2 3.73 128 22.66 27.08 18 4 3.54 100 19.95 24.39 19 4 3.26 106 13.79 26.28 20 1 2.30 90 21.18 18.85 21 0 1.74 76 15.27 13 of 9 Page 19.44 Pozoa Mean (+ s.d.) 2.00 (+ 1.49) 2.79 (+ 0.68) 100.00 (+ 15.97) 19.04 (+ 3.34) 22.92 (+ 3.67) Mann–Whitney –2.695 (0.007) –2.394 (0.017) –1.059 (0.289) –0.881 (0.387) –0.211 (0.863) U-test: Z (two- tailed signiﬁcance)

The Mann–Whitney U-test was used to assess the signiﬁcance of difference between the two species.

Downloaded from from Downloaded http://aob.oxfordjournals.org/ at World Trade Institute on September 26, 2014 26, September on Institute Trade World at Page 10 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa

160 A Total Shannon P. coriacea is broad, ranging along the Andean mountain 35 chain. Pozoa volcanica, on the other hand, is restricted to

Shannon Diversity Index the volcanic region near Volca´n Lonquimay in southern 140 Chile. Complex alternative hypotheses might be formulated, 30 obviously, to suggest that P. volcanica might have been the 120 original progenitor that survived in refugia during 25 Pleistocene glaciation, followed by derivative speciation into 100 P. coriacea and subsequent extensive range expansion north and south. The broader level of genetic variation in 20 80 P. coriacea, however, in contrast to that in P. volcanica, Total number of bands argues against this possibility (see below). The range of eco- 15 logical tolerance of P. coriacea is also much broader than 60 that of P. volcanica. The former is found in numerous habitats in and around Nothofagus and Araucaria forests, in varying

20 B 11 Private Rarity 8 types of substrate, including organic soils. Pozoa volcanica, Downloaded from * on the other hand, is restricted to open sites in the active vol- 7 canic region around Volca´n Lonquimay. Similar differences in 15 the habitat occur in other cases of progenitor–derivative spe-

6 6 Rarity Index * ciation: Layia glandulosa (progenitor species) lives on sandy 5 soils and L. discoidea (derivative) on serpentine soils 10 (Baldwin, 2005); Mimulus cupriphilus (derivative from http://aob.oxfordjournals.org/ 4 M. guttatus) grows near copper mines (MacNair et al., 1989; Macnair and Gardner, 1998). Impetus for the present project, 5 3 in fact, came from noting that P. volcanica was one of the Number of private bands 2 early colonizers of the fresh bare volcanic ash in the explosion 10 zone of the Navidad cone of Lonquimay, which erupted in 0 1 1988 (Gonza´lez-Ferra´n, 1994). The more open and uniform P. coriacea P. volcanica P. coriacea P. volcanica habitat in which P. volcanica occurs, therefore, argues for

this species being a populational derivative into a unique eco- at World Trade Institute on September 26, 2014 F IG. 4. Boxplots of AFLP data showing the median, 25 % and 75 % quartile (box) and non-outlier range in Pozoa coriacea and P. volcanica of (A) the total logical zone from P. coriacea rather than the reverse. numbers of bands and Shannon Diversity and (B) the number of private bands Populational genetic data from AFLP analyses (Fig. 5) also and Rarity Index. Numbers by the circle and asterisks represent populations argue for P. volcanica being derived from P. coriacea. First, with middle and extreme outlier data values, respectively. SplitsTree analysis places the outgroup representatives of Asteriscium and Gymnophyton within populations of P. coriacea and not in P. volcanica. Secondly, and more com- TABLE 3. Estimates of divergence and diversity based on AFLP pelling, is that the degree of genetic variation among popula- analysis of Pozoa coriacea and P. volcanica tions of P. coriacea is much greater than that of P. volcanica (see also Fig. 4). The total number of bands, number of No. of Total Percentage of Shannon private bands and the Rarity Index all support a reduced private Rarity no. of polymorphic Diversity genetic proﬁle in P. volcanica. This is what would be expected Species bands Index bands bands Index to occur with a founder effect origin of a derivative peripheral population system from a more genetically (and ecologically) Pozoa 153 6.43 355 87.19 75.86 coriacea diverse progenitor. Pozoa 51 4.92 253 60.59 47.08 volcanica Levels of genetic variation in progenitor and derivative species The genetic characteristics that a species must have to estab- lish progenitor–derivative origins are (from Crawford et al., There are three likely alternatives: (1) origin of both species 1985; Perron et al., 2000; Jaramillo-Correa and Bousquet, from a common, now extinct, ancestor; (2) origin of 2003): (1) high genetic similarity between the two species; P. coriacea from P. volcanica; or (3) origin of P. volcanica (2) less genetic variation in the derivative species; from P. coriacea. Choosing among these alternatives involves (3) absence of alleles present in the progenitor, often in low examining data from geography, ecology and patterns of frequencies, and (4) few or no unique alleles in the derivative genetic variation. The DNA sequences as a whole are not species. very informative on this question, with the exception that AFLP data from P. coriacea and P. volcanica indicate a low with ndhF-rpl32 (Fig. 3A) the monophyletic P. volcanica FST value between the species (FST ¼ 0.2019), and hence a appears more derived. high degree of genetic similarity due to a high proportion of The geography (Fig. 2) and ecology of P. coriacea and similar alleles between them. The number of private bands P. volcanica (Fig. 1C, D) suggest strongly that the latter was for P. coriacea is three times higher than that in derived from the former. The distributional range of P. volcanica (Table 3). This is concordant with the idea of Lo´pez et al. — Progenitor–derivative speciation in Pozoa Page 11 of 13

6 2 Pozoa coriacea B 7 0·01

1 4

10 5

8 Downloaded from

Asteriscium A 21 Gymnophyton http://aob.oxfordjournals.org/ Gymnophyton Asteriscium 11 94 P. coriacea 3, 5 15 94 P. coriacea 1, 2, 4, 6, 7 P. coriacea 9, 10 88 18 19 P. coriacea 8 13 98 12 P. coriacea 11 at World Trade Institute on September 26, 2014 14 20 P. volcanica 12–21 16 17 Pozoa volcanica

Nei–Li distance = 0·05

F IG. 5. Phylogenetic tree and network from the AFLP data set: (A) condensed tree; values above the branches represent bootstrap values, numbers after species name are population numbers (see Table 1); (B) SplitsTree NeighborNet analysis of AFLP data showing genetic variation within and among populations of Pozoa coriacea (squares; red lines) and P. volcanica (circles; blue lines). The scale bar is percentage distance. reduced genetic variability via recent origin of the taxon in the individuals (bottleneck), and limited gene flow from parental context of a founder effect (Purps and Kadereit, 1998). populations. This same trend of loss of genetic variation in the derivative In conclusion, P. volcanica represents a species recently species has been documented in other species pairs. Perron derived from its progenitor P. coriacea. The volcanic activity et al. (2000) and Jaramillo-Correa (2003), investigating black in and around Volcań Lonquimay has provided an opportunity spruce (Picea mariana) and red spruce (P. rubens), showed for establishment of peripheral populations from P. coriacea that the genetic diversity of the derivative species was a through dispersal into new open habitats, and subsequent di- subset of that observed in the progenitor. Gottlieb (1974) vergence in isolation. The lower level of unique alleles in found a reduced allelic diversity in the derivative Clarkia lin- P. volcanica is consistent with the hypothesis of a founder gulata in comparison with C. biloba when analysed for elec- effect. Biogeographically, it is likely that the origin of trophoretic variation specified by eight loci. Crawford and P. volcanica occurred after Pleistocene glaciation. Local Smith (1982), using allozymes, showed a decrease in genetic glaciers along the Andean chain (Ortiz-Jaureguizar and variation in the derivative species Coreopsis nuecensis in rela- Cladera, 2006), which resulted in a cooler climate, had the tion to the progenitor species C. nuecensoides. The same effect of shifting the vegetation to lower elevations, with the general trend has been documented in (progenitor species flora rebounding upwards only after the glaciers receded given first): Lasthenia minor and L. maritima (Crawford (Simpson, 1983). This may have coincided with volcanic et al., 1985), Camassia scilloides and C. angusta (Ranker and activity, so frequent along the Chilean cordillera Schnabel, 1986), Erythronium albidum and E. propullans (Gonza´lez-Ferrań, 1994), which provided even more new (Pleasants and Wendel, 1989), and Senecio nebrodensis and ecological opportunities. These events may have been respon- S. viscosus (Kadereit et al., 1995; Purps and Kadereit, 1998). sible for stimulating speciation within Pozoa as well as The causes of decline in genetic diversity in the derivative within other genera that inhabit the southern Andean mountain taxon are several, such as origin from a small number of chain. Page 12 of 13 Lo´pez et al. — Progenitor–derivative speciation in Pozoa

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