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Molecular Phylogeny and Taxonomy of Malesherbiaceae Author(s): Karla M. Gengler-Nowak Source: Systematic Botany, Vol. 28, No. 2 (Apr. - Jun., 2003), pp. 333-344 Published by: American Society of Plant Taxonomists Stable URL: http://www.jstor.org/stable/3094002 . Accessed: 07/04/2014 19:40 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . American Society of Plant Taxonomists is collaborating with JSTOR to digitize, preserve and extend access to Systematic Botany. http://www.jstor.org This content downloaded from 189.188.40.96 on Mon, 7 Apr 2014 19:40:13 PM All use subject to JSTOR Terms and Conditions

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Page 1: Gengler Nowak, 2003

Molecular Phylogeny and Taxonomy of MalesherbiaceaeAuthor(s): Karla M. Gengler-NowakSource: Systematic Botany, Vol. 28, No. 2 (Apr. - Jun., 2003), pp. 333-344Published by: American Society of Plant TaxonomistsStable URL: http://www.jstor.org/stable/3094002 .

Accessed: 07/04/2014 19:40

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

American Society of Plant Taxonomists is collaborating with JSTOR to digitize, preserve and extend access toSystematic Botany.

http://www.jstor.org

This content downloaded from 189.188.40.96 on Mon, 7 Apr 2014 19:40:13 PMAll use subject to JSTOR Terms and Conditions

Page 2: Gengler Nowak, 2003

Systematic Botany (2003), 28(2): pp. 333-344 ? Copyright 2003 by the American Society of Plant Taxonomists

Molecular Phylogeny and Taxonomy of Malesherbiaceae

KARLA M. GENGLER-NOWAK

2426 Cranford Road, Columbus, Ohio 43221 ([email protected])

Communicating Editor: Gregory M. Plunkett

ABSTRACT. Malesherbiaceae are an angiosperm family allied with Tumeraceae and Passifloraceae. The family contains 24 species in the single genus, Malesherbia, and is distributed in the arid Andes and coastal deserts of Chile, Peru, and Argentina. Although there are several morphologically cohesive groups of species in the genus, no subgenera or sections have been recognized. A phylogeny for the family was reconstructed from ITS sequence data using parsimony, implied weights, successive approximations, and maximum likelihood analyses. Parsimony, implied weights, and successive ap- proximations yielded almost identical topologies having four strongly supported clades and one weakly supported clade. Maximum likelihood analysis using model TrNef+G resulted in a topology showing the same four well supported clades, but the root moved to break the monophyly of the fifth clade. The four well-supported clades each contain morphologically similar species, and the fifth clade also shows some morphological cohesion. The five clades are morphologically divergent and may be recognized taxonomically at the level of section. Five sections, Albitomenta, Cyanpetala, Malesherbia, Parvis- tella, and Xeromontana, are described. A key to the sections is provided.

Malesherbiaceae are an angiosperm family closely related to Turneraceae and Passifloraceae (Fay et al. 1997). The distributions of the three are centered in the neotropics, but, unlike their relatives, the members of Malesherbiaceae are xerophytes restricted to the Andes mountains and the coastal desert on their western flank in Chile, western Argentina, and Peru (Fig. 1). This desert region covers more degrees of latitude than any other intensely arid desert (Trewartha 1966), and Malesherbiaceae occur over a large portion of it. The family is therefore ideal for the study of the biogeog- raphy of the entire region, which until recently has not been investigated in any detail (Rundel et al. 1991).

Malesherbiaceae exhibit a suite of morphological characters reminiscent of both Turneraceae and Passi- floraceae. The flowers of Malesherbiaceae are charac- terized by a prominent floral tube (probably of axial origin [A. Bernhard, University of Zurich, pers. comm.]) from which the perianth and a corona arise, a long androgynophore, and a persistent perianth. Un- like in Passifloraceae, the three styles are free and widely spaced upon the ovary apex. The seeds of Ma- lesherbiaceae lack the aril characteristic of the family's relatives. The leaves of the often densely pubescent Malesherbiaceae are almost always ciliate with glan- dular hairs exuding an oily, sticky fluid, and most spe- cies have stipules, which are often multiple-lobed. In addition to these morphological traits, these perennials produce cyanogenic glycosides; they are unique in that tetraphyllin A and tetraphyllin B are the dominant compounds (Spencer and Seigler 1978).

Using rbcL sequences, Fay et al. (1997) showed Ma- lesherbiaceae to be most closely related to Turneraceae, with Passifloraceae as the sister to these families. It is unknown if Malesherbiaceae and Turneraceae split be- fore Tumeraceae radiated (i.e. Tumeraceae are mono- phyletic) or if Malesherbiaceae constitute a highly

modified branch nested within Turneraceae. Given Tumeraceae's distribution in South America and Af- rica, however, it seems biogeographically unlikely that both the families are monophyletic. Raven and Axelrod (1974) contend that Tumeraceae became established on both continents during the Paleocene (65-45 mya). Ma- lesherbiaceae are almost certainly not that ancient (Gengler-Nowak 2002a), suggesting that Turneraceae may not be monophyletic.

Malesherbiaceae are little known outside of Chile. The first comprehensive treatment of Malesherbiaceae was a traditional revision by Ricardi (1967). In his treatment, Ricardi recognized 27 species in a single genus, Malesherbia R. et P. Phenetic analyses of a mor- phological complex of four species indicates that two of these species are best treated as varieties (M. gabrie- lae and M. taltalina) and that a third (M. multiflora) is indistinct from one of the varieties (Gengler-Nowak 2002b). In this study, therefore, 24 species are recog- nized. Although there appear to be several morpho- logically cohesive groups of species in the genus, Ri- cardi neither recognized nor erected sections or sub- genera recognizing this diversity.

The focus of this study is the construction of a hy- pothesis of the phylogeny of Malesherbiaceae using the internal transcribed spacer regions (ITS) of nuclear ribosomal DNA. This phylogeny is examined in light of the family's morphology. Five sections are described based upon the ITS phylogeny and the distribution of morphological traits.

MATERIALS AND METHODS

Sampling. Leaf material was collected for 39 accessions of Ma- lesherbia representing all 24 species and dried in silica gel (Table 1). When available, more than one population of each species was sampled; more extensive sampling, however, was generally not feasible due to the rarity of many of the species and/or their in- accessibility. Four species could not be located in the field; DNA

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FIG. 1. Distribution of Malesherbiaceae.

was extracted from herbarium material of these species as well as additional accessions of six other species previously collected in the field. Fresh leaves of the outgroup taxa, Piriqueta caroliniana and Turnera scabra of Tumeraceae, were collected from green- house-grown plants. This family was chosen because Tumeraceae have been implicated as the sister taxon of Malesherbiaceae (Fay et al. 1997) and because Ricardi (1967) noted that Piriqueta and Malesherbiaceae share some morphological characters, such as a corona and floral tube. Turnera is a neotropical genus closely re- lated to Piriqueta (Arbo 1995). The only other New World genera in Tumeraceae are Cuban and central American and are therefore more unlikely sister taxa.

DNA Isolation and Sequencing. Depending upon the amount of material available, total DNA extractions were carried out using a modified large-scale CTAB extraction protocol (Doyle and Doyle 1987) with subsequent purification on CsCl/ethidium bromide gradients (Palmer 1982) or a CTAB miniprep protocol after Doyle and Doyle (1987) and Cullings (1992). Miniprep-isolated DNA was not purified by CsCl gradient.

Direct, symmetrical amplification of the ITS regions and inter- vening 5.8S region was carried out using primers ITS-4 and ITS- 5 of White et al. (1990), with ITS-5 modified according to Sang et al. (1995). In a few cases, amplification of DNA from herbarium material was particularly difficult, so ITS 1 was amplified using

primers ITS-2 (White et al. 1990) and the modified ITS-5, while ITS 2 was amplified using primers ITS-3 (White et al. 1990) and ITS-4. The PCR reaction followed a hot start protocol: the initial cycle was 5 min at 95?C followed by 6 min at 72?C, during which time Taq polymerase was added to each reaction volume and the reactions capped with two drops of mineral oil. The next 30 cycles consisted of 1 min at 95?C, 1 min at 50?C, and 45 sec at 72?C. The extension segment (at 72?C) was extended by 4 sec each cycle after the first segment of 45 sec. A final extension of 5 min at 72?C completed the reaction. Amplification products were purified ei- ther in agarose gels (lx TAE) by electrophoresis and subsequent separation from the agarose using glass milk (U.S. Bioclean, Amer- sham Corp., Arlington Heights, IL) or by centrifugal filtration us- ing Ultrafree-MC filter tubes (Millipore Corp., Bedford, MA). Re- covered, clean amplification products were concentrated for se- quencing.

Manual sequencing of the purified, double-stranded ITS ampli- fication products was performed using the Sequenase version 2.0 (Amersham Corp., Arlington Heights, IL) dideoxy chain termina- tion method with forward primers ITS-5 and ITS-3 and reverse primers ITS-2 and ITS-4. The reaction protocol followed that of Sang et al. (1994). Electrophoresis of the sequences was performed in 6% acrylamide gels using wedge spacers. Gels were run at 1,500 mA until bromphenol stain migrated to the end of the gel (ap- proximately 2.5 hrs) and subsequently fixed in 10% acetic acid. They were then dried to 3-MM Whatman filter paper for 2 hrs under vacuum before exposure to Kodak XAR x-ray film for one to seven days. The ITS regions of two species, Malesherbia weber- baueri and M. turbinea, were sequenced using an automated se- quencer due to manual sequencing difficulties (Molecular Genetics Facility of the University of Georgia Research Services, Athens, GA).

Data Analyses. Boundaries of the ITS regions were deter- mined using sequences previously published for the coding re- gions of 18S, 5.8S, and 26S adjacent to the ITS regions (compiled in Torres et al. 1990). Sequences were aligned using Clustal W (Thompson et al. 1994) with a 10-point gap penalty, a transition preference, a gap extension penalty of 0.05, and a 3-point prefer- ence for identical bases. Minor manual adjustments to the com- puter alignment were made to maximize the number of invariable sites and decrease the number of indels. Most of these adjustments were made within the first 50 base pairs of ITS 1.

Analyses of the data set utilized NONA 1.6 (Goloboff 1993a), Pee-Wee 2.15 (Goloboff 1993b), and Hennig86 (Farris 1988). In par- simony analysis using NONA, 100 replicates were performed, holding 60 trees per replicate and using tree bisection-reconnec- tion. Further swapping was not necessary. These commands were also used in Pee-Wee, which optimizes the fit of the characters on the tree by their implied weights. In Hennig86, successive ap- proximations character weighting was performed after conversion to the required numerical format. IUPAC symbols for polymor- phisms were converted to unknowns by default for this process, increasing the portion of missing data to 0.68% from 0.30% of the data matrix. Gaps are treated as missing data in all three pro- grams, and polymorphic sites are accepted by NONA and Pee- Wee.

Jackknife, Bremer, and bootstrap support values were computed using Xac (Farris 1996), NONA, and PAUP*4.0b2 for Macintosh (Swofford 1999), respectively. In the calculation of the jackknife values, 10,000 replicates were performed with branch swapping and five random addition sequences per replicate. Bremer sup- ports were calculated for the consensus of the most parsimonious trees from NONA. Because Davis (1995) found that Bremer sup- port values may be inflated if all the topologies in suboptimal trees have not been searched, and because it is computationally difficult to calculate all of these trees, three separate runs were performed to determine if the results vary as more trees are searched. Un- changing support values across runs would suggest that most of the topologies have been explored and that the support values are not egregiously overestimated. In these three runs, 2,502, 5,502, and 10,502 trees up to five steps longer were calculated. Boot- straps were calculated from 5,000 replicates using "fast" step-wise

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TABLE 1. Accessions used for DNA isolation and sequencing, with GenBank No. (ITS 1/ITS 2). All vouchers are deposited at OS unless otherwise noted.

M. angustisecta Harms KMG & Refulio R. 199, AF276343/AF276396 M. ardens J. F. Macbr. KMG & Arakaki 184, AF276347/AF276400; KMG & Arakaki 185, AF276337/AF276390; KMG & Arakaki

187, AF276363/AF276416 M. arequipensis Ricardi Dillon, Sagastegui, & Santisteban 4793, AF276342/AF276395; KMG, Solas, & Cuadras 58, AF276364/

AF276417; KMG & Cuadras 182, AF276341/AF276394 M. auristipulata Ricardi KMG 61, AF276366/AF276419; KMG 65, AF276365/AF276418 M. campanulata Ricardi Ricardi, Marticorena, & Matthei 1748, AF276330/AF276383 M. densiflora Phil. KMG 115, AF276378/AF276401 M. deserticola Phil. Dillon & Dillon 6013, AF276323/AF276376; KMG 93, AF276349/AF276402 M. fasciculata D. Don KMG 151, AF276322/AF276375; Stuessy 9803, AF276321/AF276374 M. haemantha Harms Hutchinson 1278 (UC), AF276354/AF276407 M. humilis Poepp.

var. parviflora (Phil.) Ricardi KMG 106c, AF276338/AF276391; KMG 106d, AF276335/AF276388; KMG 190, AF276336/ AF276389; Marticorena, Stuessy, & Baeza 9883, AF276318/AF276371; Stuessy 9892, AF276319/AF276372

M. lactea Phil. KMG 44, AF276324/AF276377; Stuessy 9876, AF276317/AF276370 M. lanceolata Ricardi KMG 54, AF276327/AF276380; KMG 55, AF276328/AF276381; KMG 133, AF276351/AF276404; Stuessy

12809, AF276329/AF276382 M. linearifolia (Cav.) Pers. KMG 23, AF276331/AF276384; KMG 36, AF276326/AF276379; KMG 155, AF276332/AF276385 M. lirana Gay var. lirana Stuessy 9814, AF276352/AF276405 M. lirana Gay var. subglarifolia Kuntze Hjertling 6312 (A), AF276353/AF276406 M. obtusa Phil. var. obtusa KMG 119a, AF276334/AF276387 M. paniculata D. Don KMG 28, AF276325/AF276378 M. rugosa Gay var. rugosa KMG 37, AF276333/AF276386; KMG 114, AF276340/AF276393; KMG 118, AF276339/AF276392 M. scarlatiflora Gilg KMG 286, AF276360/AF276413; KMG 287, AF276359/AF276412; KMG & Romero 295, AF276357/

AF276410; KMG & Roque 350, AF276356/AF276409; KMG & Salvador P 369, AF276358/AF276411 M. splendens Ricardi KMG 188, AF276350/AF276403 M. tenuifolia D. Don KMG 191, AF276344/AF276397; KMG 192, AF276346/AF276399; KMG 197, AF276345/AF276398 M. tocopillana Ricardi Dillon & Dillon 5719, AF276320/AF276373 M. tubulosa (Cav.) J. St.-Hil. KMG & Roque 354, AF276361/AF276420; KMG & Bedoya 362, AF276362/AF276415 M. turbinea J.E Macbr. KMG & Refulio 198, AF276367/AF276420 M. weberbaueri Gilg var. weberbaueri KMG 288, AF276355/AF276408 Piriqueta caroliniana (Walter) Urban Shore 175, AF276315/AF276368 Turnera scabra Millsp. Shore 150, AF276316/AF276369

addition, which Mort et al. (2000) found to yield bootstraps com- parable to a heuristic search.

Maximum likelihood was also employed using PAUP*4.0b8 (Swofford 2001) and Modeltest 3.04 (Posada and Crandall 1998). Two accessions of Malesherbia scarlatiflora, KMG 369 and KMG 287, were removed from the data set because they were identical to a third accession of the species, KMG 350. ModelTest was imple- mented to determine which model and parameters should be ap- plied in maximum likelihood analysis using PAUP*. The resulting model and initial parameters were then used in a maximum like- lihood analysis using the "asis" option for the addition sequence, TBR, and one of the most parsimonious trees from the NONA analysis as a starting tree. The resulting tree was saved and the new parameters used in the subsequent maximum likelihood anal- ysis. This procedure was repeated until the tree topology stabi- lized. The parameters of the final analysis were then used in a more exhaustive maximum likelihood analysis using a random addition sequence with ten repetitions.

A second data set was constructed in which sites with ambig- uous alignments were deleted to determine what their effects are on the complete data set. The eight sequence segments deleted (21 sites total) each contained a short indel with more than one equal- ly plausible alignment. Parsimony analysis using NONA was per- formed as above to determine how the topology might be affected.

Because the branch between the ingroup and the outgroup is very long (104 mutations using ACCTRAN optimizations on the complete data set in PAUP*) and the root falls on one of the longer branches in the ingroup, long branch attraction could be a source of potential phylogenetic problems (Felsenstein 1978; Hendy and Penny 1989; Huelsenbeck 1997). Siddall and Whiting (1999) noted that the elimination of either of the long branches should have no

effect on the placement of the other long branch if these branches are not attracting each other. Therefore, sequences of the outgroup or the (M. linearifolia-M. paniculata) lineage from the complete and the unambiguous data sets were removed, with subsequent parsimony analysis using NONA.

RESULTS

Sequence Data. The ITS regions of the 24 species recognized by Gengler-Nowak (2002b) were success- fully amplified and sequenced. The aligned sequences were 466 bp long with 175 informative characters. Ab- solute lengths of the combined ITS 1 and ITS 2 regions ranged from 420 bp in the outgroups, Turnera scabra and Piriqueta caroliniana, to 450 bp in Malesherbia ardens (KMG 185), M. campanulata (Ricardi, Marticorena, and Matthei 1748), M. densiflora, M. lirana var. subglabrifolia, and M. rugosa (KMG 37). The shortest sequence within Malesherbiaceae was that of M. ardens (KMG 184), which had 444 bp. Within the ingroup, indels of one to three bases were scattered. A few indels in the out- groups were longer; in addition to scattered indels of one to four bases, there was one gap of 11 bases in the

outgroup not found in the ingroup. G+C content ranged from 52.2% in M. ardens (KMG 185) to 61.9% in Turnera scabra.

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Sequence divergences in Malesherbiaceae calculated with MEGA 1.0 (Kumer et al. 1993) using the Kimura 2-parameter model were greatest between all acces- sions of Malesherbia tenuifolia and M. paniculata (10.00%). Divergences were zero between M. humilis var. parviflora (Stuessy 9892) and three other accessions of that variety (KMG 106c, 106d, and 190); M. tocopil- lana and M. ardens (KMG 187), M. angustisecta and M. arequipensis (KMG 58, Dillon et al. 4793); and among M. scarlatiflora (all accessions), M. splendens, and M. zweber- baueri. Within the ingroup, only a few short sections, which were deleted for analysis of an unambiguous data set, were difficult to align due to the presence of indels. Divergences between the outgroup and ingroup ranged from 41.99% to 54.02%; divergence between Turnera scabra and Piriqueta caroliniana was 29.01%. De- spite these differences, the outgroup and ingroup se- quences could be easily aligned by eye for approxi- mately half the sequence.

Phylogeny. Malesherbiaceae were strongly sup- ported as a monophyletic clade in all phylogenetic analyses relative to the limited outgroup sampling used here. Parsimony analysis of informative charac- ters of the equally weighted data matrix yielded four shortest trees of 301 steps with a consistency index of 0.76 and retention index of 0.92 (Fig. 2). These trees differ in two respects only. The (M. ardens-M. toco- pillana) clade is sister to the (M. auristipulata-M. tur- binea) clade in two trees, whereas in the other two trees the (M. ardens-M. tocopillana) clade is sister to the (M. arequipensis-M. tenuifolia) clade. The other inconsis- tency lies in the (M. scarlatiflora-M. weberbaueri) clade. The two accessions of M. tubulosa are either most close- ly related and sister to the remaining taxa in the clade, or one of the accessions falls within the polytomy con- taining M. scarlatiflora, M. splendens, and M. weberbaueri.

Two analyses were performed using weighted data. The program Pee-Wee generated two trees of greatest fit (total fit = 1,828.9). One (301 steps) was identical to the most parsimonious trees in which the (M. ardens- M. tocopillana) clade is sister to the (M. arequipensis- M. tenuifolia) clade (Fig. 2). The other Pee-Wee tree (302 steps) differed from the first in that the two accessions of M. lactea are paraphyletic with respect to the acces- sions of M. fasciculata, rather than sister to this species.

Successive approximations character weighting was also employed. This method generated 384 trees. Like the Pee-Wee trees, the strict consensus tree also placed the (M. ardens-M. tocopillana) clade sister to the (M. arequipensis-M. tenuifolia) clade. The (M. linearifolia- M. paniculata) clade collapsed in this tree. The remain- der of the tree was identical to the consensus parsi- mony tree.

Bremer support values for the trees were constant across the three runs, although the number of trees investigated was more than quadrupled from 2,502 to

10,502. This suggests that the number of different kinds of topologies in the set of trees up to five steps longer is relatively small, indicating that the Bremer support values were not grossly overestimated.

In the consensus tree of the parsimony analyses, Ma- lesherbiaceae are broken into several well-supported clades (clades A, B, D, and E; Fig. 2) and one clade with weak support (clade C; Fig. 2). Relationships among species within these clades are poorly resolved. Composed exclusively of Peruvian and extreme north- ern Chilean species, clade E is subdivided into two clades. Clade El, with four species, is strongly sup- ported as monophyletic, but its sister clade, E2, is weakly supported. This second clade is composed of Malesherbia haemantha and three small clades; the re- lationships among these components remain unre- solved. Clade E2 collapsed in the parsimony analysis of the completely unambiguous data.

The remainder of the species is strictly Chilean. Weakly supported as sister to the largely Peruvian clade E is a relatively large, strongly supported clade of seven species (clade D; Fig. 2). The larger of the two lineages in clade D (clade D2) shows Malesherbia cam- panulata and M. lanceolata as sister taxa, but the rela- tionships among this lineage, M. lirana, M. obtusa, and M. rugosa are unresolved. Clade D2 also suffered some loss of resolution when the 21 ambiguous sites were removed (Fig. 2). The second lineage (clade Dl) con- tains only M. deserticola and M. densiflora.

The sister clade (clade C) to clades D and E contains only Malesherbia fasciculata and M. lactea, which are very weakly supported as a monophyletic group by two homoplasious characters in the NONA analysis and in one tree of the Pee-Wee analysis (Fig. 2). The Malesherbia humilis lineage (clade B) lies sister to clade C+D+E, and the (M. linearifolia-M. paniculata) line- age (clade A) is sister to the rest of the family.

The maximum likelihood analysis was completed using a data set with the two of three identical acces- sions of Malesherbia scarlatiflora removed. Analysis us- ing Modeltest indicated that the TrNef+G model (Ta- mura and Nei 1993) best fits the data. The final shape parameter derived from the iterative analyses was 1.441209. The tree resulting from the maximum like- lihood analysis (Fig. 3) shows some differences from the parsimony trees. The two accessions of M. auristi- pulata comprise a clade sister to M. turbinea, and, with- in the M. humilis lineage, two accessions group togeth- er. The (M. ardens-M. tocopillana) clade is sister to the (M. auristipulata-M. turbinea) clade.

More significantly, there are rearrangements of the relationships among the major clades due to a change in the placement of the root (Piriqueta + Turnera). The sister relationship between Malesherbia fasciculata and M. lactea is broken. Malesherbia lactea is sister to a clade containing M. fasciculata and all other accessions of Ma-

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GENGLER-NOWAK: PHYLOGENY OF MALESHERBIACEAE

Piriqueta Turnera lOG

Cyanpetala 5/100/100 M. linearifolia 5/100/3100

M. linearifolia ~9 1~~1/63/63 ] M. linearifolia

1 M. paniculata

5/100/ M. humilis 100 (B Parvistella 4/93/90 M. humilis .Q.104 Y^/ ^^M. humilis

M. humilis M. humilis

Alioet ?/ 5/ 100/100 M.fasciculata 3/81/ K Albitomenta 1/-/- | 9 M.fasciculata C

3 I 5/98/97 M. lactea 9 ,6\> 11 I M. lactea

5/96/92 I M. densifiora 5/91/ 5 11/63/70 M. deserticola D1

25/91/ 1 M. deserticola 1/ /

D 82 M. obtusa 2 6 M. lirana v. lirana

5/99/ M. lirana v. subglab. Xeromontana 97 1/-/ 1/-/53 M. rugosa

7 1 | M/-/ 5 . rugosa M. rugosa D2

2/84/85 | M. campanulata 4 11/63/77 M. lanceolata

1/-/- 1 | 1 |1/6354 M. lanceolata

1 1 I/M. lanceolata M. lanceolata M. tubulosa M. tubulosa M. scarlatiflora

_(___ 5/97/97 M. scarlatiflora 6 M. scarlatiflora E1

M. scarlatiflora M. scarlatiflora

Malesherbia 5/99/96 M. splendens Malesherb 5/99/96 ^M. weberbaueri

12 M. haemantha 2/77/82 M. auristipulata

\4 | M. auristipulata M. turbinea

1/63/60 3/95/96 M. tocopillana

M. ardens () 1 3 M. ardens

M. ardens E2 Bremer / Jackknife / Bootstrap 5/100/ M. arequipensis

M. arequipensis # mutations (ACCTRAN) 100 M. arequipensis 15 /6362M. angustisecta

1/63/62 I M. tenuifolia M. tenuifolia M. tenuifolia

FIG. 2. Consensus tree derived from the four most parsimonious trees calculated by NONA. Dashes indicate support values of less than 50. Major lineages are marked by letters (circled and on the right side) and the section names. The two heavy-type arrows indicate the two clades which collapse in the consensus tree obtained when ambiguous sites were removed and the data matrix re-analyzed in NONA. The two consensus trees are otherwise topologically identical. OG = outgroup.

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0.223 Piriqueta I 0_____.223__ ~ Turnera PG

0.139 M. lactea 0.020I 0.009 M. lactea k

____ M. fasciculata

Cyanpetala 0.002 M. linearifolia

0.502 M. linearifolia o0.002 M. paniculata

0.002 M. humilis Parvistella M. humilis

>>0.001 1- r ' --------- ^^^ -- IM. humilis 0.022 0.002 1-?00022 M. humilis

M. humilis

(P J__I 0.029 -- M. densflora [

57 0009 M. deserticola 1 0.002 0.002 M. deserticola

.0 -0.014 0 007 M. obtusa 0.005 ,<

U*U14 -- - M. lirana v. lirana

Xe/romontan 0.017 M. lirana v. subglab. Xeromontana / - M. rugosa

D2 0.017 0.002 -- M. rugosa 0-002 0.009 M. rugosa D2

0.007 M. campanulata 0.008 | M. lanceolata

0.004 M. lanceolata 0.002 M. lanceolata

0.004 M. lanceolata M. tubulosa

0.009 M. tubulosa 0.014

' M. scarlatiflora

0.002 M. scarlatiflora El 0.002 0.002 M. scarlatiflora M. splendens M. weberbaueri

Malesherbia 0.018 M. haemantha 0.70.0.028 M. auristipulata 0.028 00 0.002 M. auristipulata

0.007 M. turbinea 0.002 0.002 M. tocopillana

0.005 M. ardens 00.010 0.0116 M. ardens

M. ardens E2 0.002 M. arequipensis

M. arequipensis M. arequipensis M. angustisecta M. tenuifolia

0.002 M. tenuifolia M. tenuifolia

FIG. 3. The tree yielded by maximum likelihood analysis using model TrNef+G. For comparison, the major lineages found in the parsimony analyses are marked by letters (circled and on the right); sections are also labeled. Branch lengths are indicated below branches. Branches lacking a number have a length of zero. OG = outgroup.

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lesherbia. Malesherbia fasciculata, in turn, is paraphyletic to the remaining ingroup members. The M. humilis lin- eage is sister to the (M. linearifolia-M. paniculata) clade.

To explore the behavior of Malesherbiafasciculata and M. lactea, which appear to be a source of instability, parsimony analyses were conducted in which either one or the other species was deleted. In both analyses, the two species remained sister to clade D+E.

Parsimony analyses of the complete and unambig- uous data sets with the elimination of only the out- group taxa each yielded four topologies identical to those recovered by the original parsimony analysis of the complete data set. When only the (M. linearifolia- M. paniculata) lineage was removed, however, topolog- ical rearrangements resulted. Using the complete data set, M. fasciculata moved as sister to the remaining in- group, and the remaining major lineages and M. lactea formed a variety of relationships with each other. Us- ing the unambiguous data set, M. fasciculata was again sister to the ingroup, with M. lactea falling as sister to the remaining taxa.

DISCUSSION

As implied by the family's unique combination of morphological characteristics and in accordance with other workers' conclusions (Ricardi 1967; Takhtajan 1980; Cronquist 1981), the species of Malesherbiaceae form a monophyletic clade. The monophyly of the fam- ily, however, would be best tested using a data set in- cluding more genera in Turneraceae and a more slowly evolving gene.

The placement of the root of Malesherbiaceae may impact the formation of biogeographical inferences for the family. The parsimony analyses show the root to fall between the (M. linearifolia-M. paniculata) lineage and the rest of the family. The entire family, however, is defined by a large number of autapomorphies, and it could be argued that the placement of the root is the result of long branch attraction to the (M. linearifolia- M. paniculata) branch (Felsenstein 1978; Hendy and Penny 1989; Huelsenbeck 1997), which is long relative to most other branches within the ingroup. Siddall and Whiting (1999) noted that a pair of long branches can- not attract each other in a spurious topology if one of those branches is absent. The analyses eliminating ei- ther the outgroup or the (M. linearifolia-M. paniculata) branch are equivocal. The stability of the ingroup to- pology to the removal of the outgroup indicates that the placement of the (M. linearifolia-M. paniculata) branch relative to other ingroup taxa is not misleading. Removal of the (M. linearifolia-M. paniculata) branch, on the other hand, destabilized the relationships among the major lineages enjoying good support in the parsimony analysis and broke up the (M. fascicu-

lata-M. lactea) lineage by placing the root between M. fasciculata and the other taxa.

The lack of data concerning the relationships among the major lineages likely plays a role in these results. When the (M. linearifolia-M. paniculata) clade is re- moved, the six characters supporting the monophyly of clades B+C+D+E become synapomorphies for the family. The relationships among these major clades are supported by very few mutations, which could easily result in movement of the root and rearrangements of the major clades, especially considering the possible long-branch attraction between the outgroup branch and the rather long M. fasciculata branch. The results of sampling error (by ignoring one of the major family lineages) may also be influencing the new topology; Lecointre et al. (1993) investigated the effects of taxon sampling and found incomplete samples often yield misleading phylogenetic hypotheses. To investigate these possibilities more fully using parsimony analy- sis, a data set which resolves with greater confidence the relationships among the major lineages of Malesh- erbiaceae and the outgroups is needed.

Maximum likelihood has been suggested to be less susceptible to long branch attraction (Huelsenbeck 1997,1998; but see Siddall and Whiting 1999). Analysis of the data using maximum likelihood again implicates Malesherbia fasciculata and M. lactea as sources of to- pological instability, because the root inserts between these species, with M. lactea falling sister to the rest of the family (Fig. 3). Interestingly, removal of either of these species in parsimony analysis results in topolo- gies identical to the full parsimony analysis, suggest- ing that these taxa are not erroneously placed together by long-branch attraction (Siddall and Whiting 1999). This finding further indicates that the greatest insta- bility in the topology is due to rooting uncertainties.

Maximum likelihood and parsimony methods, then, are in disagreement as to the exact placement of the root. All analyses agree, however, that the root lies among the Chilean taxa (clades A, B, C, and D) and involves either the (M. linearifolia-M. paniculata) or (M. fasciculata-M. lactea) clade.

Although there are few characters supporting the relationships among the clades at the base of the ITS phylogeny, clades A, B, D, and E each enjoy high sup- port values (Fig. 2). These clades also are morpholog- ically distinct. The first of these clades (clade A) con- tains Malesherbia linearifolia and M. paniculata. These species have intensely blue or purple petals and sepals that are very large compared to the relatively incon- spicuous floral tube. The flowers are in panicles atop long, slender branches bearing shortly pubescent leaves. These species are native to central Mediterra- nean and semi-arid Chile.

The second lineage (clade B; Fig. 2) contains only Malesherbia humilis. This species is composed of five

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varieties (Gengler-Nowak 2002b) distributed over 11? in latitude along the coast and in the pre-Andes of Chile, with some disjunct populations reported from Province Neuquen, Argentina (37?30'S) (Hunziker and Espinar 1967). These plants, which do not grow taller than 30 cm, produce multitudes of small flowers with white or pale lavender or blue perianth parts and often greenish floral tubes. The pedicels are bractless. Stip- ules may be obsolete, and the leaves and flowers are pilose. Maximum likelihood analyses indicate that this species forms a monophyletic clade with M. linearifolia and M. paniculata. Morphologically, M. humilis is so di- vergent from the other species that there is as yet no evidence from morphology that these two lineages are very closely related.

Species adapted to montane and very dry desert habitats comprise the well-supported clade D (Fig. 2). This lineage is differentiated from others by white flowers tinted with clear yellow in the throat (with the exception of Malesherbia obtusa, which has pale blue flowers) and a pilose androgynophore (the plesio- morphic condition for the family is glabrous). The sev- en species of this clade are separated into two lineages. Malesherbia deserticola and M. densiflora (clade D1) are distinguished by having unequal, asymmetrically shaped stipules or stipules largely fused with the leaf blade and petiole. In clade D2, the stipules of the five species are obsolete or reduced to small flaps of tissue. The coronas of this clade's species are very short or reduced to a thick band of tissue at the base of the perianth.

The last clade of Chilean species in the parsimony tree contains Malesherbia fasciculata and M. lactea (clade C; Fig. 2). Molecular data weakly support their close relationship and their placement as sister to clades D + E. In the parsimony analyses excluding one or the other species, the species' sister position to clades D + E remains unchanged. The morphologies of M. fasci- culata and M. lactea are very different from other spe- cies of Malesherbia. Unlike other Malesherbia, they have entire-margined leaves lacking stipules, and usually these leaves lack prominent glandular hairs. A purple hue often tints their white flowers. Both species have long, slender branches relative to plant size, although those of M. lactea are sinuous and largely under- ground; the many long, slender branches of M. fasci- culata form a spherically shaped bush. Both species are heavily covered with white, tomentose hairs. Beyond these minor similarities, M. lactea and M. fasciculata share few other features. The internodes of the slender branches of M. fasciculata are very long, and the rather small leaves are elliptic to narrowly lanceolate. The ends of the branches bear small flowers arranged in racemes or dichasia which are then compressed into compact globes. The anthers and pollen are blue. This species is native to the pre-Andean semi-arid region of

Chile. Malesherbia lactea, which is almost a cushion spe- cies, has very short internodes and spatulate leaves, and the flowers are either solitary or in few-flowered racemes. The anthers and pollen are yellow. Its native habitat lies above 3,500 m in the Andes of Chile and Argentina. If these species are sister species, the lack of morphological and molecular evidence is probably due to rapid divergence shortly after the evolution of the lineage, perhaps in response to drastically different habitats.

The family's final major lineage (clade E; Fig. 2) is largely Peruvian, with only three species (Malesherbia tenuifolia, M. tocopillana, and M. auristipulata) in three separate clades native to extreme northern Chile. In- novations in this "Peruvian" clade are the presence of a thickened band of androecial tissue where the fila- ments become free at the apex of the androgynophore and the appearance of red and orange pigmentation. Their ovaries are generally cylindrical rather than glo- bose, and the valves of their capsules, which extend beyond and often tear open the perianth and corona at maturity, are elongated. The long, tubular shape, reddish hues, and exerted stamens and styles of the flowers of many species suggest that these may be hummingbird pollinated; hummingbirds have been observed near bushes of M. ardens (K. Gengler-Nowak, pers. obs.).

Two clades comprise the "Peruvian" lineage. Clade El (Fig. 2) is composed of species having long, tubular flowers with constricted throats and petals with pilose abaxial and adaxial surfaces. The reddish-orange or yellow flowers are arranged into dense racemes held erect on sturdy stems. All four have relatively few branches, giving a candelabra-like appearance to the plants. The ITS data failed to resolve the relationships among the four species. Malesherbia splendens and M. weberbaueri, in fact, have identical ITS sequences to M. scarlatiflora (KMG 350), even though the three species are easily distinguishable. The addition to the ITS data set of morphology or another gene, such as matK, may help elucidate the relationships among these species.

Four groups of species comprise clade E2 (Fig. 2). Malesherbia auristipulata and M. turbinea are plants with blood-red flowers having black anthers and black glan- dular hairs lining the apices of the petals and sepals. Their flowers are easily dislodged from the racemes. The dark green leaves, which have broad, lobed stip- ules, are also lined with black glandular hairs. Unlike most Peruvian species, M. auristipulata and M. turbinea have floral tubes which are wider at the throat than in the middle of the tube. Access to the throat of the flow- er, however, is still restricted by the corona, which is narrower than the floral tube throat and which forms a glossy sheath exceeding the length of the perianth. This constriction of the throat of the flower by a nar- row corona in this lineage and by a narrow perianth

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in other Peruvian species suggests that it may be func- tionally important, perhaps by promoting pollinator specificity or restricting access to the flower by some nectar robbers.

Of unknown affinities within clade E is Malesherbia haemantha, which closely resembles M. auristipulata and M. turbinea with its blood-red floral tube, black pollen and anthers, and glandular hairs lining the leaves and perianth. Its corona is also relatively long, although approximately equal to the perianth, rather than great- ly exceeding it. Unlike M. auristipulata and M. turbinea, M. haemantha's leaves are deeply pinnatifid and sub- tended by lanceolate stipules. The shared traits are un- usual for the family and suggest that the three species are closely related, but the lack of molecular evidence for such a relationship may indicate that M. haemantha split from the ancestor of M. auristipulata and M. tur- binea very early in the evolutionary history of the lin- eage. Malesherbia auristipulata and M. turbinea are found near each other on opposite sides of the Chilean-Pe- ruvian political border; M. haemantha is a narrow en- demic living about 500 km to the northwest. The dis- tribution suggests that perhaps northern populations of the species' ancestor evolved in isolation, resulting in the autapomorphies defining M. haemantha.

Malesherbia angustisecta, M. arequipensis, and M. ten- uifolia constitute a clade characterized by species hav- ing highly dissected, revolute-margined leaves covered with white, tomentose hairs. When present, the stip- ules are also highly dissected. These plants are small bushes bearing their flowers in panicles, the plesiom- orphic condition of the family. The three species are also unusual in that they largely lack the intense floral colors of other species of clade E. Malesherbia arequipen- sis and M. angustisecta have greenish-white flowers, al- though the upper portion of the style and the stigma of M. angustisecta are pink. The flowers of M. tenuifolia are pink-red when exposed to sunlight; parts of flow- ers lying on the ground or in the shade lack pigmen- tation. The presence of some pink coloration in the flowers of all three species suggests that this clade lost most color production rather than retained a plesiom- orphic condition. Malesherbia arequipensis, in addition, has lost the ring of androecial tissue at the apex of the androgynophore and the long, tubular floral tube char- acteristic of all the other members of the Peruvian clade.

The last lineage in clade E2 contains Malesherbia ar- dens and M. tocopillana, which bear their orange-red flowers in dense, long racemes held on sturdy, semi- decumbent branches. The corona is deeply and sharply toothed, and the leaves are lobed and covered with glandular hairs. The characteristics shared by these species led Rundel et al. (1991) to postulate that they are closely related. Ricardi (1967) suggested that M. tocopillana, a very rare endemic to Tocopilla, Chile

(22?05'S), is more closely allied to the shrubby species in clade El. The ITS and morphological data clearly support Rundel et al.'s (1991) assessment of the affin- ities of M. tocopillana.

Molecular and morphological data are equivocal re- garding the relationships among the lineages in clade E2. The Malesherbia ardens clade shares with the M. au- ristipulata clade a reddish corona (although the color is blood red in the M. auristipulata clade and more or- angish-red in the M. ardens clade) and the presence of a glandular hair at the apex of each petal. Supporting the closer relationship of the M. ardens clade to the M. angustisecta clade is the presence of short sepals rela- tive to the floral tube, a floral tube more than twice as long as wide, and the absence of a glandular hair at the apex of each sepal. The molecular data support either placement, depending upon the type of analysis performed. Further evidence is necessary to resolve the relationships among the clades in this diverse lineage.

The monophyly of clade E2 is weakly supported in the original parsimony and maximum likelihood anal- yses. This clade collapses in analyses of the unambig- uous data set, however, and no morphological traits have been found to unite the lineages. Low confidence for monophyly of this clade may best be viewed as support for inference of a rapid radiation culminating in a diversity of Peruvian lineages, each united by a suite of unique morphological characters.

The phylogenetic hypothesis presented here, while resolving several well-supported major clades that are morphologically distinct, lacks strong support at its base and resolution at it tips. Data are needed, there- fore, that address the relationships among the major clades and among closely related species. Use of genes that evolve more slowly than ITS could test the rela- tionships among the major clades. The inclusion of more quickly evolving genes might improve the reso- lution of relationships within the major clades.

TAXONOMIC TREATMENT

Four of the five major lineages are well defined by both morphological and molecular data. Taxonomic recognition of the lineages would emphasize the mor- phological diversity among groups of Malesherbia. Al- though the (M. fasciculata-M. lactea) lineage is not strongly supported as monophyletic in the ITS phylog- eny, it has a few defining morphological characters and will thus be recognized. Recognition at the level of sec- tion is appropriate in this genus. Not only does this avoid the creation of many small genera (and therefore new binomials), but also the possibility of further bi- nomial changes if future work establishes the phylo- genetic positions of M. fasciculata and M. lactea differ- ently than this study.

Description of Malesherbiaceae. Shrubs 3 cm to 2 m tall, semidecumbent or erect, bearing branches

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sparsely to densely pubescent with both simple and glandular hairs. Leaves alternate and simple, sessile or with a short pseudopedicel; abaxial and adaxial sur- faces sparsely to densely pubescent, margins usually lined with glandular hairs. Most species having stip- ules, these simple or divided into up to ten lobes, mar- gins lined with glandular hairs. Inflorescence a ra- ceme, panicle, or, rarely, a dichasium or solitary. Flow- ers perfect and actinomorphic, usually subtended by a pair of bracts. Floral tube 4-48 mm long and persis- tent; ten-nerved; funnelform, obconical, or campanu- late; yellow, white, red, orange, or greenish; usually densely pubescent with whitish or yellow hairs. Sepals 5 and 2.5-17 mm long; white, red, orange, yellow, dark blue or purple, light blue, violet, or greenish. Petals 5 and 2-16 mm long; white, red, orange, yellow, dark blue or purple, light blue, violet, or greenish. Corona

longer than, as long as, or shorter than perianth, some- times reduced to a raised band of tissue at base of perianth; white, light yellow, orange, or red; margin erose. Androgynophore 1.5-13.5 mm, with or without a ring of thickened tissue where joining the ovary. Sta- mens 5, free and arising from the apex of the andro- gynophore, extending beyond the throat of the floral tube and corona. Anthers yellow, blue, or black, open- ing longitudinally to release tricolporate pollen. Ovary globose, cylindrical, or conical; pubescent; having one locule and three carpels; placentation parietal and ovules many. Styles 3, free and arising from apex of ovary, extending beyond the anthers. Capsule cylin- drical, campanulate, or fusiform, tri-valved, often tear- ing perianth and corona upon dehiscence. Seeds one to many, lacking an aril, ovoid with pitted seed coat. The family contains one genus, Malesherbia.

KEY TO THE SECTIONS OF MALESHERBIA

1. Petals dark blue or purple; perianth parts as long as or longer than floral tube; plants erect ............. 1. sect. Cyanpetala 1. Petals white, yellow, red, orange, or pale blue to violet; floral tube longer than perianth parts; plants erect or semidecumbent

2. Leaf margins completely entire; leaves and stem white-tomentose; flowers solitary, many-flowered globose racemes, or, rarely, in few-flowered racemes ................................................. 2. sect. Albitomenta

2. Leaf margins not completely entire; leaf and stem pubescence hispid, pilose, or velutinous; if tomentose, leaves deeply pin- natisect or flowers tubular, campanulate, or obconical; flowers in racemes or loose panicles

3. Shrublets ('30 cm); floral tube funnelform and often tightly constricted around the androgynophore . 3. sect. Parvistella 3. Shrubs (>30 cm), if shorter, leaves finely dissected; floral tube campanulate, tubular, obconical, or broadly funnelform

and expanded around androgynophore 4. Floral tube red, orange, or yellow; if white or greenish, leaves also finely pinnatisect; androgynophore + glabrous,

usually bearing a ring of thickened tissue at the base of the ovary; ovary cylindrical or conical 4. sect. Malesherbia 4. Floral tube white, greenish, or light blue; leaves never pinnatisect; androgynophore pilose and lacking ring of thick-

ened tissue; ovary globose .......................................... 5. sect. Xeromontana

1. Malesherbia sect. Cyanpetala Gengler, sect. nov. Frutices 25-150 cm alta ramis longis gracilibus er- ectis caule brevi exorientibus; foliis lanceolatis vel pinnatisectis, inferioribus 23-120 mm longis, sti- pulis saepe lobatis; tubo floris late infundibulifor- mi colore inconspicuo; perianthio tubum floris ae- quantia vel longiore, atrocyaneo vel atropurpu- reo.-Typus hic designatus: M. linearifolia (Cav.) Pers.

Shrubs 25-150 cm tall with long, slender, erect branches arising from a short stem. Leaves lanceolate or pinnatisect, 23-120 mm long, stipules often lobed. Floral tube broadly funnelform, inconspicuously col- ored; perianth equal to or longer than the floral tube, dark blue or dark purple. The dark blue or purple hue is unique in the family, and the section's name was chosen to reflect this trait.

Native to Chile (Regions III, IV, V, VI, VII, and Me- tropolitana); in the north of its range, it is largely found in the Andean foothills. In the south, the section ranges from the coast to the foothills. Ricardi (1967) notes with some doubt the claim that M. linearifolia is native to Argentina; Hunziker and Espinar (1967) found no confirmation for its presence in that country.

Species Included-Malesherbia linearifolia (Cav.) Pers., M. paniculata D. Don.

2. Malesherbia sect. Albitomenta Gengler, sect. nov. Perennes tectus trichomatibus albis tomentosis ra- mis graciles; foliorum margine integro trichoma- tibus glandulosis vulgo deficientibus; tubo floris minus quam 1.2 cm, albo interdum tincto viola- ceo.-Typus hic designatus: M. lactea Phil.

Perennials covered with white tomentose hairs with branches slender; leaf margins entire, glandular hairs usually lacking; floral tubes less than 1.2 cm long, white and sometimes violet-tinted. The section is named for the white hairs covering both species.

Native to above 3500 m in the Andes of Chile (Re- gions II, III, and IV) and adjacent Argentina (Provinces La Rioja and San Juan). Also found in the semi-arid, pre-Andean foothills of Chilean Regions IV, V, VI, and Metropolitana.

Species Included-Malesherbia lactea Phil., M. fasciculata D. Don.

3. Malesherbia sect. Parvistella Gengler, sect. nov. Fru- ticuli erecti vulgo ramosissimi ad 30 cm alta; foliis

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pinnatisectis, lanceolatis vel oblongis, pilosis, margine ciliato trichomatibus glandulosis, infer- ioribus vulgo marcidis; stipulis interdum dificien- tibus; pedicellis ebracteatis; tubo floris 4-12 mm longis, 0.5-2.0 mm diametro (2.0-6.5 mm circum- ferentia), infundibuliformi, virello, dense piloso, fauce nunquam tincta flava; perianthio albo, cae- lesti vel violaceo; androgynophoro raro piloso.- Typus hic designatus: M. humilis Poepp.

Shrublets erect, often highly branched, reaching 30 cm in height; leaves pinnatisect, lanceolate, or oblong, pilose, margins ciliate with glandular hairs, lower leaves often withered, stipules sometimes lacking; ped- icels ebracteate; floral tube 4-12 mm long, 0.5-2.0 mm in diameter (2.0-6.5 mm in circumference), funnel- form, greenish, densely pilose, throat never tinted with yellow; perianth white, light blue, or violet; androgyn- ophore rarely pilose. The perianth parts lie perpendic- ular to the floral tube of the flowers, lending the many diminutive flowers on a plant a star-like appearance. The section's name was chosen for this feature.

Native to the coast and the pre-Andes of Chile from Santiago to Guatacondo (20056'S) (Regions II, III, IV, V, and Metropolitana). Also found in the north of Prov- ince Neuquen (37030'S) of Argentina.

Species Included-Malesherbia humilis Poepp.

4. Malesherbia sect. Malesherbia, sect. nov.- Typus: M. tubulosa (Cav.) J. St.-Hil.

Shrubs with branches erect or semidecumbent; leaves lanceolate, oblanceolate, oblongo-ovate, or deep- ly pinnatisect, densely pubescent; floral tube tubular or obconical, yellow, red, orange, or sometimes white or pale green; corona usually exceeding or equaling perianth; androgynophore usually swollen at the base of the ovary; ovary cylindrical or conical.

Native to the arid, deep inter-Andean valleys, broad valleys, coast, and Andean foothills of Peru (Depart- ments Arequipa, Ayacucho, Huancavelica, Ica, Junin, Lima, Moquegua, and Tacna). Also found in the scat- tered dry river valleys of the Atacama Desert of north- ern Chile and along the coast near Tocopilla, Chile (Re- gions I and II).

Species Included-Malesherbia angustisecta Harms, M. ardens J. F Macbr., M. arequipensis Ricardi, M. auristi- pulata Ricardi, M. haemantha Harms, M. scarlatiflora Gilg, M. splendens Ricardi, M. tenuifolia D. Don, M. to- copillana Ricardi, M. tubulosa (Cav.) J. St.-Hil., M. tur- binea J. F. Macbr., M. weberbaueri Gilg.

5. Malesherbia sect. Xeromontana Gengler, sect. nov. Frutices ramosissimi erecti vel semidecumbentes; foliis dense pubescentibus; stipulis deficientibus vel redactis vel anisomorphis; tubo floris infun- dibuliformi vel campanulato, fauce vulgo tincta

flava; corona interdum diminuta instar porcae; perianthio albo vel raro caelesti, androgynophoro piloso.-Typus hic designatus: M. densiflora Phil.

Shrubs, many-branched and erect or semidecum- bent; leaves densely pubescent; stipules lacking, re- duced, or asymmetrically shaped; floral tube funnel- form or campanulate with throat often tinted yellow; corona sometimes reduced to a ridge; perianth white or rarely pale blue; androgynophore pilose.

Distributed in montane and very dry desert habitats of Chile (Regions II, III, IV, V, and Metropolitana) and extreme western Argentina (Provinces La Rioja, Men- doza, Neuquen, and San Juan). The section's name re- flects the habitats of its constituent species.

Species Included-Malesherbia campanulata Ricardi, M. densifora Phil., M. deserticola Phil., M. lanceolata Ricardi, M. lirana Gay, M. obtusa Phil., M. rugosa Gay.

ACKNOWLEDGEMENTS. I thank D. J. Crawford, A. Wolfe, K. Pickett, and T. Stuessy for their comments, help, and advice and S. Barrett and M. Cruzan for providing plant material for the out- groups. I am grateful to J. Wenzel for use of his copies of the programs NONA, Pee-Wee, and Hennig86 and his many hours of assistance with these programs and to J. Freudenstein for help with Latin. The comments of Leigh Johnson, a second reviewer, and Gregory Plunkett greatly improved the manuscript, for which I thank them. I am indebted to the faculty and staff of the follow- ing institutions: Universidad de Concepci6n, Chile; the Museo Na- cional de Historia Natural in Santiago, Chile; the Universidad de La Serena, Chile; the Museo de Historia Natural "Javier Prado" in Lima, Peru; the Universidad Nacional de San Agustin in Arequipa, Peru; the Universidad Nacional de San Cristobal de Huamanga in Ayacucho, Peru; the Museo Contisuyo in Moquegua, Peru; and the Universidad Nacional del Centro del Peru in Huancayo, Peru. I thank the Instituto Nacional de Recursos Naturales (INRENA) of Peru for permission to collect and export specimens from that country. I further thank the following herbaria for the loan of spec- imens of Malesherbia for this study: A, CONC, CTES, F, GH, MO, MOL, NY, SGO, UC, ULS, US, and USM. This research, conducted at The Ohio State University, was funded by NSF Doctoral Dis- sertation Improvement Grant DEB-9623496, the Tinker Foundation, the Flora of Chile project, the Beatley Herbarium Grant, and Sigma Xi Grants-in-Aid of Research.

LITERATURE CITED

ARBO, M. M. 1995. Tumeraceae Parte I. Piriqueta. Flora Neotropica. Monograph 67. The New York Botanical Garden, Bronx, New York.

CRONQUIST, A. 1981. An integrated system of classification offlowering plants. Columbia University Press, New York, NY.

CULLINGS, K. W. 1992. Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1: 233-240.

DAVIS, J. I. 1995. A phylogenetic structure for the Monocotyledons, as inferred from chloroplast DNA restriction site variation, and a comparison of measures of clade support. Systematic Botany 20: 503-527.

DOYLE, J. J. and J. L. DOYLE. 1987. A rapid DNA isolation proce- dure for small quantities of fresh leaf material. Phytochemical Bulletin 19: 11-15.

FARRIS, J. S. 1988. Hennig86 version 1.5. MS-DOS program, Port Jefferson, New York.

2003] 343

This content downloaded from 189.188.40.96 on Mon, 7 Apr 2014 19:40:13 PMAll use subject to JSTOR Terms and Conditions

Page 13: Gengler Nowak, 2003

SYSTEMATIC BOTANY

. 1996. Parsimony jackknifing outperforms neighbor-joining. Cladistics 12: 99-124.

FAY, M. E, S. M. SWENSEN and M. W. CHASE. 1997. Taxonomic affinities of Medusagyne oppositifolia (Medusagynaceae). Kew Bulletin 52: 111-120.

FELSENSTEIN, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Systematic Zoology 27: 401-410.

GENGLER-NOWAK, K. M. 2002a. Reconstruction of the biogeo- graphical history of Malesherbiaceae. The Botanical Review 68: 171-188.

--. 2002b. Phenetic analyses of morphological traits in the Ma- lesherbia humilis complex (Malesherbiaceae). Taxon 51: 281- 293.

GOLOBOFF, P. A. 1993a. NONA, version 1.6. MS-DOS program. Published by the author, San Miguel de Tucuman, Argentina.

--. 1993b. Pee-Wee version 2.15. MS-DOS program. Published by the author, San Miguel de Tucuman, Argentina.

HENDY, M. D. and D. PENNY. 1989. A framework for the quanti- tative study of evolutionary trees. Systematic Zoology 38: 297-309.

HUELSENBECK, J. P. 1997. Is the Felsenstein zone a fly trap? Sys- tematic Biology 46: 69-74. . 1998. Systematic bias in phylogenetic analysis: is the Strep-

siptera problem solved? Systematic Biology 47: 519-537. HUNZIKER, A. T. and L. A. ESPINAR. 1967. Nota aclaratoria sobre

las Malesherbiaceae argentinas y una clave para su identifi- caci6n. Kurtziana 4: 83-86.

KUMER, S., K. TAMURA, and M. NEI. 1993. MEGA: Molecular evo- lutionary genetics analysis, version 1.0. The Pennsylvania State University, University Park, PA.

LECOINTRE, G., H. PHILIPPE, H. L. V. LE, and H. LEGUYADER. 1993. Species sampling has a major impact on phylogenetic infer- ence. Molecular Phylogenetics and Evolution 2: 205-224.

MORT, M. E., P. S. SOLTIS, D. E. SOLTIS and M. L. MABRY. 2000. Comparison of three methods for estimating internal support on phylogenetic trees. Systematic Biology 49: 160-170.

PALMER, J. D. 1982. Isolation and structural analysis of chloroplast DNA. Nucleic Acids Research 10: 167-186.

POSADA, D. and K. A. CRANDALL. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817-818.

RAVEN, P. H. and D. I. AXELROD. 1974. Angiosperm biogeography and past continental movements. Annals of the Missouri Bo- tanical Garden 61: 539-673.

RICARDI, M. 1967. Revisi6n taxon6mica de las Malesherbiaceas. Gayana Botanica. No. 16:1-139.

RUNDEL, P. W., M. O. DILLON, B. PALMA, H. A. MOONEY, S. L. GULMON, and J. R. EHLERINGER. 1991. The phytogeography and ecology of the coastal Atacama and Peruvian deserts. Aliso 13: 1-49.

SANG, T., D. J. CRAWFORD, S.-C. KIM, and T E STUESSY. 1994. Ra- diation of the endemic genus Dendroseris (Asteraceae) on the Juan Fernandez Islands: evidence from sequences of the ITS regions of nuclear ribosomal DNA. American Journal of Bot- any 81: 1494-1501. , D. J. CRAWFORD, and T. E STUESSY. 1995. ITS sequences

and the phylogeny of the genus Robinsonia (Asteraceae). Sys- tematic Botany 20: 55-64.

SIDDALL, M. E. and M. E WHITING. 1999. Long-branch abstrac- tions. Cladistics 15: 9-24.

SPENCER, K. C. and D. S. SEIGLER. 1978. Cyanogenic glycosides of Malesherbia. Biochemical Systematics and Ecology 13: 23-24.

SWOFFORD, D. L. 1999. PAUP*. Phylogenetic Analysis Using Par- simony (* and Other Methods). Version 4. Sinauer Associates, Sunderland, MA.

2001. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4. Sinauer Associates, Sun- derland, MA.

TAKHTAJAN, A. L. 1980. Outline of the classification of flowering plants (Magnoliophyta). Botanical Review 46(3): 225-359.

TAMURA, K. and M. NEI. 1993. Estimation of the number of nucle- otide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512-526.

THOMPSON, J. D., D. G. HIGGINS, and T. J. GIBSON. 1994. Clustal W: improving the sensitivity of progressive multiple se- quence alignment through sequence weighting, position spe- cific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680.

TREWARTHA, G. T. 1966. The Earth's problem climates. The Uni- versity of Wisconsin Press, Madison, Wisconsin.

TORRES, R. A., M. GANAL, and V. HEMLEBEN. 1990. GC balance in the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes. Journal of Molecular Evolution 30: 170-181.

WHITE, T. J., T. BRUNS, S. LEE, and J. TAYLOR. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 in PCR protocols: A guide to methods and amplifications, eds. M. Innis, D. Gelfand, J. Sninsky, and T. White. San Diego, CA: Academic Press.

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