Kenneth J. Sytsma

Angiosperm phylogeny: 17 genes, 640 taxa

PREMISE OF THE STUDY Recent analyses employing up to five genes have provided numerous insights into angiosperm phylogeny, but many relationships have remained unresolved or poorly supported. In the hope of improving our understanding of angiosperm phylogeny, we expanded sampling of taxa and genes beyond previous analyses. METHODS We conducted two primary analyses based on 640 species representing 330 families. The first included 25260 aligned base pairs (bp) from 17 genes (representing all three plant genomes, i.e., nucleus, plastid, and mitochondrion). The second included 19846 aligned bp from 13 genes (representing only the nucleus and plastid). KEY RESULTS Many important questions of deep-level relationships in the nonmonocot angiosperms have now been resolved with strong support. Amborellaceae, Nymphaeales, and Austrobaileyales are successive sisters to the remaining angiosperms (Mesangiospermae), which are resolved into Chloranthales + Magnoliidae as sister to Monocotyledoneae + [Ceratophyllaceae + Eudicotyledoneae]. Eudicotyledoneae contains a basal grade subtending Gunneridae. Within Gunneridae, Gunnerales are sister to the remainder (Pentapetalae), which comprises (1) Superrosidae, consisting of Rosidae (including Vitaceae) and Saxifragales; and (2) Superasteridae, comprising Berberidopsidales, Santalales, Caryophyllales, Asteridae, and, based on this study, Dilleniaceae (although other recent analyses disagree with this placement). Within the major subclades of Pentapetalae, most deep-level relationships are resolved with strong support. CONCLUSIONS Our analyses confirm that with large amounts of sequence data, most deep-level relationships within the angiosperms can be resolved. We anticipate that this well-resolved angiosperm tree will be of broad utility for many areas of biology, including physiology, ecology, paleobiology, and genomics.

Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: Insights from an eight-locus plastid phylogeny

PREMISE Bromeliaceae form a large, ecologically diverse family of angiosperms native to the New World. We use a bromeliad phylogeny based on eight plastid regions to analyze relationships within the family, test a new, eight-subfamily classification, infer the chronology of bromeliad evolution and invasion of different regions, and provide the basis for future analyses of trait evolution and rates of diversification. METHODS We employed maximum-parsimony, maximum-likelihood, and Bayesian approaches to analyze 9341 aligned bases for four outgroups and 90 bromeliad species representing 46 of 58 described genera. We calibrate the resulting phylogeny against time using penalized likelihood applied to a monocot-wide tree based on plastid ndhF sequences and use it to analyze patterns of geographic spread using parsimony, Bayesian inference, and the program S-DIVA. RESULTS Bromeliad subfamilies are related to each other as follows: (Brocchinioideae, (Lindmanioideae, (Tillandsioideae, (Hechtioideae, (Navioideae, (Pitcairnioideae, (Puyoideae, Bromelioideae))))))). Bromeliads arose in the Guayana Shield ca. 100 million years ago (Ma), spread centrifugally in the New World beginning ca. 16-13 Ma, and dispersed to West Africa ca. 9.3 Ma. Modern lineages began to diverge from each other roughly 19 Ma. CONCLUSIONS Nearly two-thirds of extant bromeliads belong to two large radiations: the core tillandsioids, originating in the Andes ca. 14.2 Ma, and the Brazilian Shield bromelioids, originating in the Serro do Mar and adjacent regions ca. 9.1 Ma.

Salvia (Lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of Salvia and tribe Mentheae

Salvia, with over 900 species from both the Old and New World, is the largest genus in the Lamiaceae. Unlike most members of the subfamily Nepetoideae to which it belongs, only two stamens are expressed in Salvia. Although the structure of these stamens is remarkably variable across the genus, generally each stamen has an elongate connective and divergent anther thecae, which form a lever mechanism important in pollination. In a preliminary investigation of infrageneric relationships within Salvia, the monophyly of the genus and its relationship to other members of the tribe Mentheae were investigated using the chloroplast DNA regions rbcL and trnL-F. Significant conclusions drawn from the data include: Salvia is not monophyletic, Rosmarinus and Perovskia together are sister to an Old World clade of Salvia, the section Audibertia is sister to subgenus Calosphace or the monotypic Asian genus Dorystaechas, and the New World members of section Heterosphace are sister to section Salviastrum. Owing to the non-monophyly of Salvia, relationships at the next clearly monophyletic level, tribe Mentheae, were investigated.

Origin, adaptive radiation and diversification of the Hawaiian lobeliads (Asterales: Campanulaceae)

The endemic Hawaiian lobeliads are exceptionally species rich and exhibit striking diversity in habitat, growth form, pollination biology and seed dispersal, but their origins and pattern of diversification remain shrouded in mystery. Up to five independent colonizations have been proposed based on morphological differences among extant taxa. We present a molecular phylogeny showing that the Hawaiian lobeliads are the product of one immigration event; that they are the largest plant clade on any single oceanic island or archipelago; that their ancestor arrived roughly 13 Myr ago; and that this ancestor was most likely woody, wind-dispersed, bird-pollinated, and adapted to open habitats at mid-elevations. Invasion of closed tropical forests is associated with evolution of fleshy fruits. Limited dispersal of such fruits in wet-forest understoreys appears to have accelerated speciation and led to a series of parallel adaptive radiations in Cyanea, with most species restricted to single islands. Consistency of Cyanea diversity across all tall islands except Hawai i suggests that diversification of Cyanea saturates in less than 1.5 Myr. Lobeliad diversity appears to reflect a hierarchical adaptive radiation in habitat, then elevation and flower-tube length, and provides important insights into the pattern and tempo of diversification in a species-rich clade of tropical plants.

Congruence Versus Phylogenetic Accuracy: Revisiting the Incongruence Length Difference Test

Phylogenies inferred from independent data partitions usually differ from one another in topology despite the fact that they are drawn from the same set of organisms (Rodrigo et al., 1993). Some topological differences are due to sampling error or to the use of inappropriate phylogenetic models. These types of topological incongruence do not have their origin in genealogical discordance, i.e., differences between phylogenies underlying the respective data partitions (Baum et al., 1998). Incongruence that is not due to genealogical discordance can often be addressed by modifying the model used in phylogenetic reconstruction (Cunningham, 1997b), and combining data is an appropriate way of dealing with random topological differences that are attributable to sampling error. However, other topological differences, e.g., those arising from lineage sorting (Maddison, 1997; Avise, 2000) and hybridization (Dumolin-Lapegue et al., 1997; Rieseberg, 1997; McKinnon et al., 1999; Avise, 2000), reflect genealogical discordance between the data partitions. Most systematists consider data partitions to be combinable if and only if they are not strongly incongruent with one another (Sytsma, 1990; Bull et al., 1993; Huelsenbeck et al., 1996; Baum et al., 1998; Johnson and Soltis, 1998; Thornton and DeSalle, 2000; Yoder et al., 2001; Barker and Lutzoni, 2002; Buckley et al., 2002). Systematists who follow this prior agreement or conditional combination approach to analyzing multiple data partitions (Bull et al., 1993; Huelsenbeck et al., 1996; Johnson and Soltis, 1998) evaluate incongruence using tests such as the incongruence length difference (ILD) test (Farris et al., 1994, 1995) or other tests of taxonomic congruence (Templeton, 1983; Kishino and Hasegawa, 1989; Larson, 1994; Shimodaira and Hasegawa, 1999) before deciding whether the partitions should be analyzed in combination. Data that exhibit strong incongruence are then analyzed separately or under assumptions that minimize incongruence (Cunningham, 1997b). In their article “Failure of the ILD to determine data combinability for slow loris phylogeny,” Yoder et al. (2001) critiqued the ILD test based on the observation that it will sometimes identify data partitions as incongruent when in fact those partitions combine to produce an accurate estimate of organismal phylogeny. They described the ILD test as a failed test of data combinability, maintaining that the presumed accuracy of trees inferred from combined data indicates the congruence of the data partitions. We have two objections to their argument (2001:421) that “the ILD [should] never be used as a test of data partition combinability.” First, what Yoder et al. described as a flaw in the ILD test as applied to their data, i.e., an apparent inverse relationship between phylogenetic accuracy and data partition congruence as measured by the ILD test, turns out to be an artifact of analysis. There is in fact a bimodal relationship between congruence and accuracy: as either data partition is upweighted, homoplasy in the combined data set is swamped by homoplasy within the upweighted data partition, reducing the significance of the ILD test. At the same time, the topology of the combined analysis shifts to reflect the topology of the upweighted data partition. This phenomenon is predictable and can be accounted for in the analysis (Dowton and Austin, 2002). Second, Yoder et al.’s expectation that ILD test results should predict the phylogenetic accuracy of the combined data analysis is unreasonable. The ILD test is used to evaluate the null hypothesis that characters that make up two or more data partitions are drawn at random from a single population of characters, i.e., a population of characters that reflects a single phylogeny and a single set of evolutionary processes (Farris et al., 1995). Because accuracy of trees derived from a data set depends on many factors other than congruence among data partitions, the ILD test cannot be used to directly address questions related to phylogenetic accuracy. Genealogically discordant data can be combined to yield accurate phylogenies, whereas data that are congruent (both genealogically concordant and homogeneous in underlying evolutionary process) can be combined to yield phylogenies that do not accurately represent organismal history (Cunningham, 1997a). A damaging critique of the ILD test would have to appeal to criteria other than

Phylogeny of Capparaceae and Brassicaceae based on chloroplast sequence data

Capparaceae and Brassicaceae have long been known to be closely related families, with the monophyly of Capparaceae more recently questioned. To elucidate the relationship between Brassicaceae and Capparaceae as well as to address infrafamilial relationships within Capparaceae, we analyzed sequence variation for a large sampling, especially of Capparaceae, of these two families using two chloroplast regions, trnL-trnF and ndhF. Results of parsimony and likelihood analyses strongly support the monophyly of Brassicaceae plus Capparaceae, excluding Forchhammeria, which is clearly placed outside the Brassicaceae and Capparaceae clade and suggest the recognition of three primary clades-Capparaceae subfamily (subf.) Capparoideae, subf. Cleomoideae, and Brassicaceae. Capparaceae monophyly is strongly contradicted with Cleomoideae appearing as sister to Brassicaceae. Two traditionally recognized subfamilies of Capparaceae, Dipterygioideae and Podandrogynoideae, are embedded within Cleomoideae. Whereas habit and some fruit characteristics demarcate the three major clades, floral symmetry, stamen number, leaf type, and fruit type all show homoplasy. Clades within Capparoideae show a biogeographical pattern based on this sampling. These results are consistent with several alternative classification schemes.

CHLOROPLAST DNA EVOLUTION AND PHYLOGENETIC RELATIONSHIPS IN CLARKIA SECT. PERIPETASMA (ONAGRACEAE)

Restriction‐site analysis of chloroplast DNA in Clarkia sect. Peripetasma (Onagraceae) was done to test previously proposed phylogenetic models. One hundred nineteen restriction‐site mutations were identified among the nine species using 29 restriction enzymes, and these were used to construct rooted most parsimonious trees (Wagner and Dollo). A chloroplast DNA evolutionary clock could not be statistically rejected. Branch points of this tree were statistically tested by Felsensteins bootstrap method. This tree 1) provided an unambiguous and detailed genealogical history for the section, 2) verified a previous partial phylogenetic model for the section based on gene duplications and differential silencing, 3) provided details of the phylogenetic model not inferred or expected based on morphology and reproductive isolation, and 4) indicated that morphology evolves at markedly different rates within and between lineages in the section.

ANCIENT VICARIANCE OR RECENT LONG-DISTANCE DISPERSAL? INFERENCES ABOUT PHYLOGENY AND SOUTH AMERICAN-AFRICAN DISJUNCTIONS IN RAPATEACEAE AND BROMELIACEAE BASED ON ndhF SEQUENCE DATA

Rapateaceae and Bromeliaceae each have a center of diversity in South America and a single species native to a sandstone area in west Africa that abutted the Guayana Shield in northern South America before the Atlantic rifted. They thus provide ideal material for examining the potential role of vicariance versus long‐distance dispersal in creating amphiatlantic disjunctions. Analyses based on ndhF sequence variation indicate that Rapateaceae and Bromeliaceae are each monophyletic and underwent crown radiation around 41 and 23 Ma, respectively. Both exhibit clocklike sequence evolution, with bromeliads evolving roughly one‐third more slowly than rapateads. Among rapateads, the divergence of west African Maschalocephalus dinklagei from its closest South American relatives implies that Maschalocephalus resulted via long‐distance dispersal 7 Ma, not ancient continental drift; only its sandstone habitat is vicariant. Rapateads arose first at low elevations in the Guayana Shield; the earliest divergent genera are widespread along riverine corridors there and, to a lesser extent, in Amazonia and the Brazilian Shield. Speciation at small spatial scales accelerated 15 Ma with the invasion of high‐elevation, insular habitats atop tepuis. Among bromeliads, Pitcairnia feliciana diverges little from its congeners and appears to be the product of long‐distance dispersal ca. 12 Ma. Brocchinia/Ayensua and then Lindmania are sister to all other bromeliads, indicating that the Guayana Shield was also the cradle of the bromeliads. Three lineages form an unresolved trichotomy representing all other bromeliads: (1) Till andsioideae, (2) Hechtia, and (3) a large clade including remaining genera of Pitcairnioideae and all Bromelioideae. The last includes a clade of pitcairnioid genera endemic to the Guayana and Brazilian Shields; a xeric group (Abromeitiella/Deuterocohnia/Dyckia/Encholirium/Fosterella) from southern South America and the southern Andes, sister to Pitcairnia; and Andean Puya, sister to Bromelioideae, with many of the latter native to the Brazilian Shield. Both Rapateaceae and Bromeliaceae appear to have arisen at low elevations in the Guayana Shield, experienced accelerated speciation after invading dissected mountainous terrain, and undergone long‐distance dispersal to west Africa recently. Bromeliad acquisition of key adaptations to drought (e.g., CAM photosynthesis, tank habit, tillandsioid leaf trichomes) 17 Ma appears to have coincided with and help cause the centripetal invasion of drier, more seasonal regions beyond the Guayana Shield, resulting in a wider familial range and dominance of the epiphytic adaptive zone. Geology, past and present climate, and proximity to South America help account for both families occurring in nearly the same area of Africa. We present a new classification for Rapateaceae, including a new tribe Stegolepideae, a new subfamily Monotremoideae, and revisions to tribe Saxofridericieae and subfamily Rapateoideae.

Clades, Clocks, and Continents: Historical and Biogeographical Analysis of Myrtaceae, Vochysiaceae, and Relatives in the Southern Hemisphere

Some of the most interesting but still most contentious disjunct biogeographical distributions involve Southern Hemisphere tropical and warm temperate families. The PHMV clade of Myrtales includes four families (Psiloxylaceae, Heteropyxidaceae, Myrtaceae, and Vochysiaceae) that exhibit a number of these biogeographical patterns. The related Psiloxylaceae and Heteropyxidaceae are small families restricted in distribution to the recent volcanic Mascarene Islands to the east of Madagascar and to southeast Africa, respectively. Myrtaceae are found on three major Gondwanan regions (South America, Australasia, and Africa). Because the New World taxa are almost exclusively fleshy fruited, it is unclear whether the family distribution is a classic Gondwanan vicariance pattern or results from one or more long‐distance dispersal events over ocean barriers. The Vochysiaceae represent one of a handful of families with amphi‐Atlantic distributions vigorously argued to support both long‐distance dispersal over the Atlantic and vicariance of western Gondwanan biota by Atlantic seafloor spreading. Molecular phylogenetic relationships, fossil dating of nodes, and penalized likelihood rate smoothing of maximum likelihood trees were employed for a Myrtales‐wide analysis using rbcL and ndhF and an analysis of the PHMV analysis using ndhF and matK. The results indicate that the PHMV differentiated during the late Cretaceous. The African lineage of Vochysiaceae is nested within a South American clade and probably arose via long‐distance dispersal in the Oligocene at a time when the Atlantic had already rifted 80 m.yr. at the equatorial region. The African/Mascarene Island families, most closely related to Myrtaceae, differentiated during the late Eocene, with subsequent but recent long‐distance dispersal from Africa to the Mascarenes. Myrtaceae show a rapid differentiation of a basal, paraphyletic subf. Leptospermoideae in Australasia. Fleshy‐fruited taxa (subf. Myrtoideae) are not monophyletic. Vicariance of a widespread warm temperate Southern Hemisphere distribution is likely in explaining the South American–Australasian disjunction, with subsequent dispersal events between the two and to Africa and the Mediterranean basin.

Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data

Despite intensive morphological and molecular studies of Onagraceae, relationships within the family are not fully understood. One drawback of previous analyses is limited sampling within the large tribe Onagreae. In addition, the monophyly of two species-rich genera in Onagreae, Camissonia and Oenothera, has never been adequately tested. To understand relationships within Onagraceae, test the monophyly of these two genera, and ascertain the affinities of the newly discovered genus Megacorax, we conducted parsimony and maximum likelihood analyses with rbcL and ndhF sequence data for 24 taxa representing all 17 Onagraceae genera and two outgroup Lythraceae. Results strongly support a monophyletic Onagraceae, with Ludwigia as the basal lineage and a sister-taxon relationship between Megacorax and Lopezia. Gongylocarpus is supported as sister to Epilobieae plus the rest of Onagreae, although relationships within the latter clade have limited resolution. Thus, we advocate placement of Gongylocarpus in a monogeneric tribe, Gongylocarpeae. Most relationships within Onagreae are weakly resolved, suggesting a rapid diversification of this group in western North America. Neither Camissonia nor Oenothera appears to be monophyletic; however, increased taxon sampling is needed to clarify those relationships. Morphological characters generally agree with the molecular data, providing further support for relationships.