Phylogeny and generic limits in the Niemeyera complex of New Caledonian Sapotaceae: evidence of multiple origins of the anisomerous flower

Molecular Phylogenetics and Evolution 49 (2008) 909–929

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Phylogeny and generic limits in the Niemeyera complex of New Caledonian Sapotaceae: evidence of multiple origins of the anisomerous flower Ulf Swenson a,*, Porter P. Lowry II b,c, Jérôme Munzinger d, Catarina Rydin e, Igor V. Bartish f a

Department of Phanerogamic Botany, Swedish Museum of Natural History, P. O. Box 50007, SE-104 05 Stockholm, Sweden Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299, USA c Département Systématique et Evolution, Muséum National d’Histoire Naturelle, Case Postal 39, 57 rue Cuvier, 75231 Paris CEDEX 05, France d US084, Laboratoire de Botanique et d’Ecologie Appliquées, Centre IRD de Nouméa, B. P. A5 98848 Nouméa Cedex, New Caledonia e Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland f Université de Rennes 1, Unité Mixte de Recherche CNRS 6553 « Ecobio »: Ecosystèmes—Biodiversité—Evolution, Campus de Beaulieu, Bâtiment 14A, 263 Avenue du Général Leclerc, 35042 Rennes Cedex, France b

a r t i c l e

i n f o

Article history: Received 14 May 2008 Revised 4 September 2008 Accepted 12 September 2008 Available online 2 October 2008 Keywords: Ancestral reconstruction Anisomery Character evolution Conservation Classification IUCN

a b s t r a c t Traditional generic limits within the ‘‘Niemeyera complex” (Sapotaceae: Chrysophylloideae) in Australia and New Caledonia do not correspond to natural groups. We analyzed nuclear (ETS, ITS) and chloroplast (trnH–psbA, trnS–G) sequence data, and 42 morphological characters, using a near-complete taxon sampling. The resulting phylogeny provides a new generic framework where Leptostylis and Sebertia are monophyletic, Niemeyera is recognized as a small genus confined to Australia, and the circumscriptions of Achradotypus and Pycnandra require significant modification. This framework allows about 20 recently discovered species to be described in appropriate genera and assessed for their conservation status. Evolutionary changes in two widely used characters, anisomerous flowers and the number of stamens inserted opposite each corolla lobe, have occurred multiple times. There is no evidence that anisomery originated through hybridization as suggested in other groups. Instead, the two characters are closely coupled and often mutually exclusive. The diagnostic value of morphological characters varies considerably; for example, the presence, absence, and type of malpighiaceous hairs convey more phylogenetic information than many traditionally used features. Criteria and options for a new generic classification are discussed, and a reconstruction of the hypothesized ancestor that likely colonized New Caledonia in the Oligocene is presented. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction The southwest Pacific island of New Caledonia has a diverse and highly endemic flora, with an estimated 3270 native vascular plant species, about 75% of which occur nowhere else (Jaffré et al., 2004; Lowry et al., 2004). This fact provides the principal argument for recognizing the territory as a biodiversity hotspot and a global conservation priority (Myers et al., 2000). One important component of the island’s native flora is the plant family Sapotaceae in the order Ericales (Anderberg et al., 2002), which is New Caledonia’s eighth most speciose group, comprising 84 species distributed in 16 genera sensu Jaffré et al. (2004). Despite their numerical importance and the fact that they are represented in nearly every native vegetation type, Sapotaceae have long suffered from seemingly intractable taxonomic problems involving the delimitation of both genera and species. Understanding the evolution of such diverse groups and circumscribing taxa in a way that reflects underlying * Corresponding author. Fax: +46 08 5195 4221 E-mail address: [email protected] (U. Swenson). 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.09.022

phylogenetic relationships is essential for biologists, ecologists, and conservationists alike, and is critically important for identifying conservation priorities and implementing sound natural resource management policies. This is especially true in places such as in New Caledonia, where large portions of the remaining natural vegetation are increasingly at risk due to mining, fire, urban sprawl, invasive species and other threats (Jaffré et al., 1998; Jaffré, 2005; Meyer et al., 2006). Over the last several years we have undertaken a series of studies aimed at clarifying the taxonomy and phylogeny of Sapotaceae in New Caledonia and neighboring areas (Bartish et al., 2005; Swenson et al., 2007a,b; Munzinger and Swenson, in press). Here we report on the results of a phylogenetic analysis focused on those members of the family that lack staminodes and frequently have anisomerous flowers, the ‘‘Niemeyera complex”, a monophyletic group in the subfamily Chrysophylloideae (Swenson et al., 2007a). Chrysophylloideae are one of three subfamilies of Sapotaceae (Swenson and Anderberg, 2005). They are composed of shrubs and medium understorey to giant canopy trees of rainforests in Africa, Australasia, and South America. Chrysophylloideae

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currently include about 600 species in 28 recognized genera but the numbers are likely to increase as a result of ongoing phylogenetic and taxonomic research (Swenson et al., 2008). Several recent molecular phylogenetic studies based on nuclear ribosomal (ITS) sequences data and morphology have examined relationships among the Australasian members of the subfamily (Bartish et al., 2005; Swenson et al., 2007a,b; Triono et al., 2007). These studies provide strong support for five well-defined lineages whose flowers bear staminodes, i.e., Beccariella, Pichonia, Planchonella, Sersalisia, and Van-royena (Swenson et al., 2007a). All these genera were accepted by Aubréville (1964, 1967) but were later (except Pichonia) included in Pouteria by Pennington (1991), an interpretation that is not supported by the molecular studies (Swenson and Anderberg, 2005; Swenson et al., 2007a, 2008). Among Australasian Chrysophylloideae is the monophyletic Niemeyera complex, forming the sister to Planchonella (Swenson et al., 2007a, 2008). However, generic delimitation within this complex remains unclear. The group’s diagnostic characters are obscure and numerous as yet undescribed taxa cannot be assigned to any of the currently accepted genera. In New Caledonia, the Niemeyera complex is composed of Leptostylis (8 spp.) and Pycnandra (12 spp.) (Vink, 1957; Aubréville, 1967; Pennington, 1991; Swenson and Anderberg, 2005) and a number of small segregate genera accepted by Aubréville (1967) including ‘‘Corbassona” (2 spp., but see Section 2.1 below), Niemeyera (1 sp.), Ochrothallus (9 spp.), Sebertia (2 spp.), and Trouettia (2 spp.). In Australia the complex is represented by at least five species of Niemeyera. The complex came about when Pennington (1991) transferred several (but not all) elements of Aubréville’s (1967) segregate genera to Niemeyera. Recent studies have demonstrated that the Niemeyera species restricted to Australia form the sister group to all taxa endemic to New Caledonia, that ‘‘Corbassona”, Leptostylis, and Sebertia are monophyletic (each with strong support), and that Ochrothallus and possibly also Pycnandra are polyphyletic (Bartish et al., 2005; Swenson et al., 2007a). However, resolution within the New Caledonian clade has been limited in the studies conducted to date, with many lineages collapsing into a basal polytomy, especially when morphological characters are introduced into the analyses. The current overall interpretation is that the clade originated in New Caledonia following a single dispersal and colonization event with subsequent rapid radiation. Like the rest of Sapotaceae, the Niemeyera complex comprises woody taxa ranging in habit from shrubs to medium-sized and large trees (Fig. 1). Most species have their leaves clustered at the tips of branches and flowers are born below in fascicles, sometimes in dense inflorescences. All members are characterized by the absence of staminodes, indistinguishable stigmatic areas, and the presence of a fruit with a single seed that has plano-convex cotyledons and no endosperm (Swenson et al., 2007a). Some of the genera have traditionally been recognized by the presence of anisomerous flowers in which the number of sepals differs from the number of petals. The number of stamens inserted opposite each corolla lobe is another key character that has been used within the group (Vink, 1957; Aubréville, 1967; Pennington, 1991). Aubréville (1967) used various combinations of features to distinguish between his small segregate genera, including the position of stamen insertion within the corolla tube and the form (length and width) of the seed scar. For instance, he recognized Ochrothallus based on the presence of five sepals, 7–10 corolla lobes, and a single stamen inserted in the corolla tube orifice opposite each corolla lobe. By contrast, Pennington (1991) placed Ochrothallus in synonymy under Niemeyera, but left the majority of its species unassigned to genus (Govaerts et al., 2001). Leptostylis is characterized by opposite leaves, four sepals, 4–10 corolla lobes, and a single stamen inserted opposite each corolla lobe. Although Pycnandra exhibits a great deal of floral variation, species have

alternate leaves, five (or rarely six) sepals, 5–10 corolla lobes, and most often two stamens inserted opposite each corolla lobe. However, the generi-type Pycnandra benthamii has red flowers (rather than white, as in most taxa) and 3–4 stamens opposite each corolla lobe. Niemeyera sensu Pennington (1991) also has alternate leaves, five sepals, and 5–10 spreading corolla lobes, but a single stamen inserted opposite each corolla lobe. However, Swenson et al. (2007a) demonstrated that species of Niemeyera from Australia form a clade that is sister to all taxa in New Caledonia. They are characterized by five-merous flowers, anthers not flexible on their connectives, and globose fruits. Given these differing views on generic circumscription, it seems appropriate to reconsider the diagnostic value of traditionally used characters, in particular the number of sepals versus corolla lobes and the number of stamens inserted opposite each corolla lobe. At least four different flower types can be recognized based on combinations of these characters: isomerous or anisomerous flowers, respectively, with either one or two stamens opposite each corolla lobe (Fig. 2). In light of the results of our recent molecular studies, however, it is unclear whether the distribution of these characters corresponds to phylogenetic relationships or whether, instead, parallel or convergent evolution has taken place within the Niemeyera complex. The anisomerous flower is an unusual feature among eudicotyledons, although it has been reported in groups such as Trientalis (Myrsinaceae) and several genera of Araliaceae. Trientalis is a small genus comprising four species confined to the northern temperate zone characterized by five sepals and often seven petals. Manns and Anderberg (2005) studied the phylogenetic relationships among the herbaceous members of Myrsinaceae and postulated that Trientalis, based on its incongruent placement in the molecular analyses, may have originated through hybridization followed by polyploidization. Anisomerous flowers with as many as 20 petals also occur in species historically assigned to araliaceous genera such as Gastonia, Munroidendron, Reynoldsia and Tetraplasandra (Philipson, 1970, 1979; Lowry, 1990), although recent studies have shown that these groups are not monophyletic as currently circumscribed (Plunkett et al., 2001, 2004; Costello and Motley, 2007). Within Sapotaceae, anisomerous flowers are also found in several other genera including Nesoluma, Magodendron, and Omphalocarpum (Swenson and Anderberg, 2005). Nesoluma, now included in Sideroxylon (Smedmark and Anderberg, 2007), is a small Pacific genus of three species whose flowers have 4 or 5 sepals and usually 7–10 corolla lobes. Smedmark and Anderberg’s (2007) molecular study indicated that Nesoluma probably originated through a hybridization event that took place between 43.0 and 36.6 million years ago involving a maternal lineage that is now confined to Africa and a paternal lineage that currently occurs only in America. The apparent hybrid origin of anisomery in such groups suggests that it may be of interest to explore whether this distinctive feature has arisen in a similar manner among New Caledonian Sapotaceae. Sapotaceae were treated more than four decades ago in the first volume of Flore de la Nouvelle Calédonie et Dépendances (Aubréville, 1967), at which time 80 species in 16 genera were listed. Several of these taxa were known and/or described only from flowering material (Ochrothallus) or sterile collections (Pycnandra paniensis). Since then, eleven additional species of Planchonella have been described (Swenson et al., 2007b; Munzinger and Swenson, in press) and several others will be published shortly, including three new species of Beccariella and two of Pichonia. Within the Niemeyera complex an additional 20 new species have been identified to date, but we have chosen to refrain from describing them until generic boundaries have been clarified. This is an important task, because many of the novelties are rare and/or occur in threatened environments, and are therefore in need of assessment using the IUCN Red List criteria (IUCN, 2001). These criteria are critical as they are used

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Fig. 1. Variation in morphology and habit within the Niemeyera complex (Sapotaceae) in New Caledonia. (A and B) Ochrothallus blanchonii, a shrub confined to maquis on ultramafic soils, (C and D) Pycnandra sp., an undescribed understorey tree growing on ultramafic soils, (E and F) Pycnandra sp., a recently discovered shrub from moist cloud forest in the northeast, (G) Pycnandra benthamii, a canopy tree growing on schist, (H) Trouettia lissophylla, a shrub from the maquis, (I) Leptostylis goroensis, a shrub found among serpentine rocks in a small area near Goro (southwest). Photos: Jérôme Munzinger (A–D, F–I) and Pete Lowry (E).

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Fig. 2. Types of flowers in the Niemeyera complex (Sapotaceae). (A) Isomerous (five-merous), (B) isomerous with two stamens opposite each corolla lobe, (C) anisomerous with one stamen opposite each corolla lobe, (D) anisomerous with two stamens opposite each corolla lobe. Drawings: Emma Hultén.

by New Caledonian environmental services as a key reference for determining which plants are threatened and for developing programs to ensure their protection (Munzinger et al., 2008). The present study aims to investigate phylogenetic relationships within the Niemeyera complex using Bayesian inference and parsimony jackknife analysis of DNA sequence data and morphology. We are particularly interested in trying to improve resolution in the phylogeny of the group and in clarifying generic delimitations, an essential step for undertaking taxonomic revisions and describing the many new taxa that have been identified. By using both nuclear and chloroplast sequence data, we also wish to investigate whether the anisomerous flower found in members of the Niemeyera complex is the result of a single evolutionary event or has multiple origins within the group, and whether hybridization may have been involved. A more fully resolved phylogenetic tree could be used to map character state changes, evaluate the diagnostic value of several traditionally used features, such as the anisomerous flower and the number of stamens, and identify previously neglected characters that may be useful for developing an improved taxonomy for the Niemeyera complex. 2. Materials and methods 2.1. Nomenclature We follow the subfamilial classification of Swenson and Anderberg (2005). The checklist of Sapotaceae (Govaerts et al., 2001), which uses Pennington’s (1991) classification, includes a full list of published names in the family. However, since Pennington’s system does not entirely convey natural groups, we herein follow the generic concepts adopted by Aubréville (1967), which are used for the sixteen undescribed taxa included in our sample that have been given provisional names (in quotation marks). Species with two stamens inserted opposite each corolla lobe are placed in Pycnandra and those with alternate leaves, an anisomerous flower, and a single stamen per corolla lobe are placed in Ochrothallus. Several taxa, however, are difficult to place and are therefore listed simply by collector and collection number (Table 1). One nomenclatural problem is pertinent to the generic name Corbassona. When Aubréville (1967) described this genus he overlooked the fact that the generi-type of Trouettia (T. leptoclada) is conspecific with the type he assigned to his new genus Corbassona (C. deplanchei), thus making Corbassona a heterotypic synonym of Trouettia. Since one purpose of the present study is to investigate phylogenetic relationships and test the generic concept sensu Aubréville, we have chosen to use the name ‘‘Corbassona” as originally defined by Aubréville, but place it in quotation marks. 2.2. Taxon sampling A total of 56 accessions representing 53 terminal taxa were used in this study (Table 1). These include the type species of all

genera recognized to date in the Niemeyera complex (except Leptostylis) and nearly all of the described species in the group (Table 2). Eight taxa for which material is unavailable (Leptostylis gatopensis, L. longiflora, L. micrantha, L. multiflora, Pycnandra decandra, P. paniensis, Ochrothallus wagapensis, and Trouettia heteromera) were not included in our study. All previous studies have shown that Leptostylis is monophyletic (see above), but the four unsampled taxa are rare and we have not been able to relocate them. Similarly, the two unsampled Pycnandra species grow in remote areas and have not been re-collected since 1978 and 1981, respectively, and their herbarium material is not useable for molecular studies. Extensive efforts to relocate O. wagapensis, which was last collected 30 years ago, were unsuccessful and we fear that this distinctive species may be extinct due to loss of habitat. Vink (1957) described two additional species of Pycnandra (P. gatopensis and P. elegans), although they were subsequently included within P. decandra by Aubréville (1967). However, material (including the type) of P. decandra from Ile Art, located off the northern tip of the main island of Grande Terre, differs morphologically from other collections currently assigned to the species, suggesting that more than one entity may be involved. Three accessions of P. fastuosa were included in our analysis because there has been some question of whether this taxon, as currently circumscribed, is monophyletic. Outgroup taxa include five species of Planchonella, a genus that is sister to the Niemeyera complex (Swenson et al., 2007a, 2008). We also sampled four Australian species of Niemeyera, which comprise a monophyletic group sister to the members of the complex occurring on New Caledonia. These served as additional outgroup taxa. The phylogenetic trees resulting from our analyses were rooted on the branch between Planchonella and the Niemeyera complex. 2.3. Nuclear data Molecular sequences of nuclear ribosomal ITS1 and ITS2 (including complete 5.8S and parts of 18S and 26S) have proven to be informative for phylogenetic analyses in Sapotaceae (Bartish et al., 2005; Swenson et al., 2007a,b). For the present study we include 38 previously published sequences to which we have added another 18 sequences obtained following the protocol for DNA extraction, amplification, and sequencing described by Bartish et al. (2005). Taxa, vouchers, GenBank accessions and primers used are reported in Tables 1 and 3. We sought to include additional nuclear and chloroplast markers. From the nucleus we derived sequences from the external transcribed spacer (ETS), which has proven to be useful for phylogenetic analyses, especially in combination with ITS (Baldwin and Markos, 1998; Markos and Baldwin, 2001; Ekenäs et al., 2007; Harbaugh and Baldwin, 2007). The ETS fragments were amplified using the primers 18S-ETS (Baldwin and Markos, 1998), Ast-1 and Ast-8 (Markos and Baldwin, 2001), and Sap-1 (50 -CGT ACT TGA GCG TGT TGG TGT-30 ), designed by Mattias Myrenås (Swedish

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Table 1 Taxa, voucher information, and GenBank accessions included in this phylogenetic study of the Niemeyera complex (Sapotaceae, Chrysophylloideae) in Australasia. Species of Planchonella were used as outgroups and Australian Niemeyera as additional outgroups. All vouchers, if not stated differently, are from New Caledonia. Two vouchers marked with an asterisk () were used for ETS. Generi-types follow Aubréville (1967) and are indicated in bold. Informal names are given in quotation marks. Sequences published here have the prefix EU. Taxon

Voucher

ITS

ETS

trnH–psbA

trnS–G

‘‘Corbassona deplanchei” (Baill.) Aubrév. ‘‘Corbassona intermedia” (Baill.) Aubrév. Leptostylis filipes Benth. Leptostylis goroensis Aubrév. Leptostylis grandifolia Vink Leptostylis petiolata Vink Leptostylis ‘‘sclerophyllea” Munzinger & Swenson Niemeyera balansae (Baill.) Aubrév. Niemeyera chartacea (F. M. Bailey) C. T. White Niemeyera prunifera (F. Muell.) F. Muell. Niemeyera whitei (Aubrév.) L. W. Jessup Niemeyera ‘‘Ford2429” Ochrothallus blanchonii Aubrév. Ochrothallus francii (Guillaumin & Dubard) Guillaumin Ochrothallus gordoniifolius (S. Moore) Aubrév. Ochrothallus litseiflorus Guillaumin Ochrothallus multipetalus (Vink) Aubrév. Ochrothallus sarlinii Aubrév. Ochrothallus schmidii Aubrév. Ochrothallus sessilifolius (Pancher & Sebert) Pierre ex Guillaumin Ochrothallus ‘‘Munzinger696” Ochrothallus ‘‘Munzinger1394” Planchonella eerwah (F. M. Bailey) P. Royen Planchonella kaalaensis Aubrév. Planchonella luteocostata Munzinger & Swenson Planchonella obovata (R. Br.) Pierre Planchonella sphaerocarpa (Baill.) Dubard Pycnandra benthamii Baill. Pycnandra carinocostata Vink Pycnandra chartacea Vink Pycnandra comptonii (S. Moore) Vink Pycnandra controversa (Guillaumin) Vink Pycnandra elegans Vink Pycnandra fastuosa (Baill.) Vink Pycnandra fastuosa (Baill.) Vink Pycnandra fastuosa (Baill.) Vink Pycnandra gatopensis Vink Pycnandra griseosepala Vink Pycnandra kaalaensis Aubrév. Pycnandra neocaledonica (S. Moore) Vink Pycnandra vieillardii (Baill.) Vink Pycnandra ‘‘Munzinger2615” Pycnandra ‘‘Munzinger2618” Pycnandra ‘‘Munzinger2624” Pycnandra ‘‘Munzinger2885” Pycnandra ‘‘Munzinger3135” Pycnandra ‘‘Swenson597” ‘‘Sapotaceae Munzinger1438” ‘‘Sapotaceae Munzinger1717” ‘‘Sapotaceae Munzinger2622” ‘‘Sapotaceae Munzinger2786” ‘‘Sapotaceae Munzinger2963” ‘‘Sapotaceae Swenson615” Sebertia acuminata Pierre ex Baill. Sebertia gatopensis (Guillaumin) Aubrév. Trouettia lissophylla (Pierre ex Baill.) Aubrév.

Munzinger 978 (NOU, MO, P, S) Cochereau s.n. (NOU) Webster & Hildreth 14665 (P) Munzinger 2288 (NOU, P, S) Munzinger & Oddi 2121 (NOU, MO, P, S) MacKee 12746 (P); Swenson & Munzinger 714 (S) Veillon 8117 (NOU, P) Munzinger et al. 1451 (NOU, MO, P, S) Australia: Bartish & Jessup 5 (S) Australia: Jessup 5238 (S) Australia: Floyd s.n. (S) Australia: Ford 2429 (S) Munzinger et al. 2576 (NOU, S) Munzinger 965 (NOU, MO, P) Swenson & Munzinger 726a (S) MacKee 17085 (P, S); MacKee 16651 (P, S) Munzinger & Swenson 2993 (S) Munzinger 1860 (NOU, P, S) McPherson & Munzinger 18106 (NOU, MO, P, S)

AY552120 AY552119 AY552135 DQ154052 DQ154053 AY552134 AY552136 AY552123 DQ154057 DQ154058 AY552137 EF025089 DQ154059 AY552117 EU661433 DQ154060 EU661434 EU661435 AY552116

EU661380 EU661381 EU661382 EU661383 EU661384 EU661385 EU661386 EU661387 NS NS EU661388 EU661389 EU661390 EU661391 EU661392 EU661393 EU661394 EU661395 EU661396

EU661499 EU661500 DQ344113 EU661501 EU661502 EU661503 EU661504 EU661505 EU661506 DQ344120 EU661507 EU661508 EU661509 DQ344121 EU661510 EU661511 EU661512 EU661513 EU661514

EU661451 EU661452 DQ344410 EU661453 EU661454 EU661455 EU661456 EU661457 EU661458 DQ344417 EU661459 EU661460 EU661461 DQ344418 EU661462 EU661463 EU661464 EU661465 EU661466

McPherson & Munzinger 18176 (MO, P) Munzinger 696 (MO, NOU, P, S) Munzinger 1394 (NOU, MO, P, S) Australia: Floyd s.n. (S) Jaffré 3505 (NOU) Munzinger et al. 2375 (NOU, P, S) Taiwan: Chung & Anderberg 1166 (S) Tronchet et al. 389 (MO, P) Munzinger, Létocart & Gateblé 2228 (NOU, P, S) McPherson & Munzinger 18091 (MO, P, S) Munzinger & Swenson 3059 (NOU, P, S) Lowry, McPherson & Le Borgne 5780A (MO, NOU, S) Lowry, McPherson & Le Borgne 5787 (MO, NOU, P, S) Munzinger, Lowry & Létocart 2031 (MO, NOU, P, S) Munzinger, Suprin & Carriconde 1281 (MO, NOU, P) Munzinger et al. 1694 (MO, NOU, P, S) Munzinger, Pignal & Lowry 2027 (NOU, P, S) Swenson, Munzinger & Butin 700b (MO, NOU, P, S) Swenson, McPherson & Mouly 627 (MO, NOU, P, S) Munzinger & Labat 2599 (NOU, S) Tronchet, Munzinger & Oddi 426 (MO, NOU, P, S) Dumontet, Zongo & Maituku s.n. (NOU) Munzinger et al. 2615 (NOU, P, S) Munzinger et al. 2618 (NOU, P, S) Munzinger et al. 2624 (NOU, P, S) Munzinger, Pillon & Butin 2885 (NOU, P, S) Munzinger et al. 3135 (NOU, P, S) Swenson, McPherson & Mouly 597 (NOU, S) Munzinger et al. 1438 (MO, NOU, P, S) Munzinger et al. 1717 (MO, NOU, S) Munzinger et al. 2622 (NOU, P, S) Munzinger & Blaffart 2786 (NOU, P, S) Munzinger et al. 2963 (MO, NOU, P, S) Swenson, McPherson & Mouly 615 (S) Munzinger 1006 (MO, NOU, P) Munzinger & Létocart 2067 (MO, NOU, P, S) Munzinger 1913 (NOU)

AY552118 AY552161 AY552133 AY552147 AY552105 EF025099 DQ154076 AY552139 EU661436 AY552132 EU661437 AY552131 AY552126 DQ154091 AY552122 AY552121 EU661438 EU661439 AY552128 EU661440 AY552129 EU661441 EU661442 EU661443 EU661444 EU661445 EU661446 AY552127 AY552159 EU661447 EU661448 EU661440 EU661450 AY552110 AY552124 DQ154092 DQ154095

EU661397 EU661398 EU661399 EU661400 NS EU661401 EU661402 EU661403 EU661404 EU661405 EU661406 EU661407 EU661408 EU661409 EU661410 EU661411 EU661412 EU661413 EU661414 EU661415 EU661416 EU661417 EU661418 EU661419 EU661420 EU661421 EU661422 EU661423 EU661424 EU661425 EU661426 EU661427 EU661428 EU661429 EU661430 EU661431 EU661432

EU661515 EU661516 EU661517 DQ344132 DQ344126 EU661518 DQ344139 DQ344146 EU661519 EU661520 EU661521 DQ344145 EU661522 EU661523 EU661524 EU661525 EU661526 EU661527 EU661528 EU661529 EU661530 EU661531 EU661532 EU661533 EU661534 EU661535 EU661536 EU661537 EU661538 EU661539 EU661540 EU661541 EU661542 EU661543 EU661544 EU661545 EU661546

EU661467 EU661468 EU661469 DQ344429 DQ344423 EU661470 DQ344437 DQ344444 EU661471 EU661472 EU661473 DQ344443 EU661474 EU661475 EU661476 EU661477 EU661478 EU661479 EU661480 EU661481 EU661482 EU661483 EU661484 EU661485 EU661486 EU661487 EU661488 EU661489 EU661490 EU661491 EU661492 EU661493 EU661494 EU661495 EU661496 EU661497 EU661498

Museum of Natural History), a primer that partially overlaps Ast-1. The PCR was slightly modified from Bartish et al. (2005) as follows: 95 °C for 5 min, followed by 4 cycles of 95 °C for 30 s, 57 °C for 30 s, 70 °C for 115 s, 4 cycles of 95 °C for 30 s, 55 °C for 30 s, 70 °C for 115 s, and 32 cycles of 95 °C for 30 s, 53 °C for 30 s, 70 °C 115 s followed by 70 °C for 8 min. Purified products were sequenced using an ABI3130xl Automated DNA Sequencer (Applied Biosystems, Foster City, California, USA). Since it is known that ribosomal DNA can occur in multiple copies in a genome, ETS sequences were carefully checked for double

GenBank accession

peaks in the proof reading procedure because the presence of several copies may indicate a taxon of hybrid origin. Three samples (Pycnandra ‘‘Swenson597”, P. chartacea, and ‘‘Sapotaceae Munzinger2786”) yielded partly unreadable sequences. These PCR products were cloned into plasmids using the TOPO TA for Sequencing Cloning Kit (Invitrogen, California, USA) following the manufacture’s instructions. Ten, four, and ten clones, respectively, were amplified and sequenced as described above. The cloned sequences were clear and a single strand was sequenced using the Ast-1 primer.

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Table 2 Comparison of Aubréville’s (1967), Pennington’s (1991), and the classification suggested herein, together with the number of accepted species for each classification. The number of sampled taxa is given in the far right column where bold numbers indicate that the generi-type was included and the values in parentheses indicate the number of undescribed taxa. ‘‘Corbassona” sensu Aubréville (1967) is a heterotypic synonym of Trouettia, and is used in quotation marks for the purpose of this study. Aubréville’s classification

Accepted species

Pennington’s classification

Accepted species

Petals versus sepals

Anthers per corolla lobe

Suggested classification

Accepted species

Number of sampled (and undescribed) taxa

Achradotypus ‘‘Corbassona” Leptostylis Niemeyera Ochrothallus Pycnandra Sapotaceae indet Sebertia Trouettia

— 2 8 2 9 12 — 2 2

— Niemeyera Leptostylis Niemeyera Niemeyera Pycnandra — Niemeyera Niemeyera

— — 8 20 — 12 — — —

An/-isomerous Isomerous Anisomerous An-/isomerous Anisomerous An-/isomerous An-/isomerous Isomerous Isomerous

Two (or one) One One One One Two to four One One One

Achradotypus (Clade H) Ochrothallus? (Clades B + C + D) Leptostylis (Clade G) Niemeyera (Clade A1) Ochrothallus? (Clades B + C + D) Pycnandra (Clade E) various genera Sebertia (Clade F) Ochrothallus? (Clades B + C + D)

14–15 16 10 4–5 16 11

14 2 5 (1) 5 (1) 8 (2) 18 (6) (6) 2 1

Table 3 List of primers used in the phylogenetic study of the Niemeyera complex (Sapotaceae). * Primer designed by Mattias Myrenås, Swedish Museum of Natural History. DNA region

Primer

Reference/sequence 50 –30

ETS ETS ETS ITS rpoB–trnCGCA

18S–ETS Ast-1, Ast-8 Sap-1 18SF, 26RN rpoB, trnCGCAR

trnHGUG–psbA trnDGUC–trnTGGU trnSGCU–trnGUUC

trnHGUG, psbA trnDGUC, trnTGGU trnSGCU, trnGUUC

Baldwin and Markos (1998) Markos and Baldwin (2001) CGT ACT TGA GCG TGT TGG TGT* Rydin et al. (2004) Ohsako and Ohnishi (2000), Shaw et al. (2005) Hamilton (1999) Shaw et al. (2005) Shaw et al. (2005)

2.4. Chloroplast data Previous molecular phylogenetic studies of Sapotaceae using chloroplast markers have found low sequence variation. The coding ndhF locus has limited use at the family level (Anderberg and Swenson, 2003) and the noncoding locus trnL–F was shown to be even less informative than ndhF in the closely related family Lecythidaceae (Mori et al., 2007). Results from the noncoding intergenic spacers trnH–psbA, trnC–petN, petN–psbM, and psbM–trnD at the subfamily level have been mixed (Smedmark et al., 2006). Shaw et al. (2005) pointed out that while the number of phylogenetic studies using noncoding chloroplast data is increasing, most rely on too few regions. In order to facilitate the use of additional markers, they conducted a comparative study on the relative usefulness of 21 regions. Based on their findings, we carried out a pilot study to estimate the amount of information in the four regions that were found by Shaw et al. (2005) to provide on average the greatest number of informative characters across angiosperms. These include the intergenic spacers trnDGUC–trnTGGU, trnSGCU–trnGUUC, rpoB–trnCGCA and trnSUGA–trnfMCAU. In addition, we investigated the usefulness of trnH–psbA. Initially, we sequenced these regions for ten species from different genera of Sapotaceae sensu Aubréville (1967) and evaluated the phylogenetic information in each region. Primers specified by Shaw et al. (2005) were used for amplification of the above mentioned regions except for trnSGCU–trnGUUC (Table 3). The suggested primers for this latter region are in part identical to those for another region (trnSUGA–trnfMCAU) described by Demesure et al. (1995) and we found that primers for trnS–trnG in fact amplified the trnS–trnfM region. To avoid possible errors and confusion we instead selected other primers for the trnS–trnG spacer designed by Hamilton (1999). We used a standard protocol: 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 120 s, followed by 72 °C for 8 min. For the trnD–trnT region, we used the same basic protocol but with an annealing temperature of 45 °C and running only 30 cycles.

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2.5. Alignment and gap coding Assembly and alignment of sequences from the four selected markers were performed in four partitions following Bartish et al. (2005) and using the similarity criterion (Simmons, 2004). Inferred gaps in both the nrDNA and cpDNA datasets were coded manually as additional binary characters following the method of Simmons and Ochoterena (2000). 2.6. Morphological data A total of 42 morphological characters (Appendix A) were scored for the 56 terminals (Appendix B). Nearly all these characters were used by Swenson et al. (2007a) and are discussed therein; those employed for the first time in the present study are discussed in Appendix A. Additional morphological data were gathered from herbarium material deposited at MO, NOU, P, and S (abbreviations follow Holmgren et al., 1990). Flowers and fruits were boiled in Copenhagen mixture (70 ml ethanol, 29 ml distilled water, 1 ml glycerol) in a microwave oven and later examined under a stereomicroscope. Some features could not be coded for a few poorly known species due to lack of adequate material, and if character states in a given taxon were indistinguishable, they were coded as polymorphic. Character states were ordered, as far as possible, so that the outgroup (Planchonella) would be assigned a value of ‘‘0”. Character coding and analyses follow Swenson et al. (2007a). For some analyses the morphological characters were combined with the molecular dataset (see below). We used MacClade 4.0 (Maddison and Maddison, 2000) to trace selected morphological characters on the Bayesian mayority-rule consensus tree generated from the molecular dataset. We originally selected a suite of traditionally used diagnostic characters, including the number of sepals versus petals (char. 13) and the number of stamens inserted opposite each corolla lobe (char. 20). Seed scar width (char. 38) appeared to be of potential interest and was therefore mapped even though we were unable to code this character for 14 of the 47 ingroup accessions included in our study. We mapped nine additional characters that either have been traditionally used or recently identified as being potential diagnostic (Swenson et al., 2007a); see Appendix A for further information. 2.7. Phylogenetic analyses The ITS and ETS loci are part of the same transcription unit and their sequences therefore cannot be considered as independent datasets, but instead should be used to augment each other (Baldwin and Markos, 1998). Combining the plastid loci trnH–psbA and trnS–G is justified by the fact that they are a single coalescent gene sensu Doyle (1995). Preliminary jackknife analyses (as described below) were performed separately for nrDNA and cpDNA sequence

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data to assess whether they would yield congruent topologies. Because the cpDNA dataset resulted in a poorly-resolved phylogeny in which only two groups received jackknife support of 90% or more and no obvious incongruence could be detected, we chose to combine the nrDNA and cpDNA partitions into a single dataset in line with a growing trend (e.g., Soltis et al., 2000; Richardson et al., 2001; Schönenberger et al., 2005; Lehtonen and Myllys, 2008) to use a total evidence approach (Lecointre and Deleporte, 2005) in phylogenetic research. The combined molecular dataset was then analyzed separately and simultaneously with the morphological dataset. Bayesian analysis (Rannala and Yang, 1996; Yang and Rannala, 1997) was used to search for better-resolved phylogenies. We examined the relative fit of various models of nucleotide substitution for the data from both fragments of cpDNA (trnH–psbA and trnS–G) and from the two different partitions of nuclear loci (ITS and ETS) to identify the best performing model in each separate partition of the total data. Our selection was based on the Akaike Information Criterion (AIC) as implemented in Model Test 3.6 (Posada and Crandall, 1998). The results of these analyses were used to create input data for MrBayes (Ronquist and Huelsenbeck, 2003), so that for each gene partition the settings used were the closest available to the best performing model. Indels were included in our phylogenetic reconstructions and treated as ‘‘morphological” characters (Lewis, 2001), with the absence of an indel coded as ‘‘0” and presence as ‘‘1”. To allow separate estimation of substitution parameters for each region of DNA and to enable the inclusion of binary data, we decoupled parameter estimation across the datasets. Two datasets, one comprising DNA sequences only, and another combining data from DNA and morphological characters, were prepared following this approach. Bayesian searches were based on 1–1.5 million generations (depending on the efficiency of parameter convergence in analyses of different data) with Markov Chain Monte Carlo starting from random trees and flat priors (default). We used four chains in all analyses, of which three were heated (temperatures from 0.05 to 0.2) and one was cold. Trees were sampled every 100th generation, providing 10001–15001 trees per run. We assumed Markov chains were stationary when the log-likelihood values reached a stable equilibrium (Huelsenbeck and Ronquist, 2001) and standard deviations decreased to less than 0.02. Majority-rule consensus phylograms and posterior probabilities (PP) for nodes were assembled from all post burn-in sampled trees. Phylogenetic reconstructions for each of the two datasets were estimated using 3 or 4 independent runs to confirm that they converged on similar stationary parameter estimates. Posterior probabilities of nodes were used to infer statistical support for clades. Values below 0.85 are considered as weak, 0.85–0.94 as moderate, and 0.95–1.00 as strong. Jackknife analyses (Farris et al., 1996) were performed, as implemented in PAUP 4.0 (Swofford, 2002), on the two datasets. The settings used in these analyses were: 1000 jackknife replicates with a single random addition sequence, TBR branch swapping, saving a maximum of 1000 trees, collapsing branches if minimum length is zero, and steepest descent not in effect. The fraction of characters excluded by jackknifing was set to 37%. Parsimony jackknifing values (JK) below 50% are not reported; group frequencies of 50–74% are considered as weak, 75–89% as moderate, and 90– 100% as strong support. These intervals differ from the posterior probabilities, which yield higher numerical values for a given amount of support when compared with nonparametric bootstrapping and/or jackknifing (Erixon et al., 2003; Simmons et al., 2004). We investigated Bremer support (Bremer, 1988) for certain groups, an approach that defines the number of extra steps necessary to collapse a group found in the strict consensus. Also, PAUP provides an option that makes it possible to impose a topological constraint that is (or is not) compatible with monophyly of a de-

sired group. We used a heuristic search of 100 stepwise random additions of taxa, TBR branch swapping, and saving multiple trees. 3. Results 3.1. Data Our pilot study indicated that two noncoding cpDNA regions, trnH–psbA and trnS–trnG, were potentially useful for phylogenetic analysis of the taxa being examined here. Two other regions, rpoB– trnC and trnD–trnT, were nearly invariable throughout the whole sequence and thus less informative, so they were not further pursued. Shaw et al. (2005) indicated that trnS–trnG is one of the most phylogenetically informative regions among angiosperms and potentially much more useful than trnH–psbA for resolving relationships at low taxonomic levels. Our analysis, however, yielded few informative characters in the trnS–G sequences, with information provided by gaps rather than from nucleotide substitutions. The entire cpDNA dataset contains 1368 characters with only 15 (0.01%) informative sites as compared to a total of 17 gaps. One portion of the trnH–psbA region was too ambiguous to align and was therefore excluded prior to analysis. By contrast, ETS (441 base pairs in length) proved to be very useful, with 103 informative characters (23.3%) and 12 coded gaps. Moreover, when ITS and ETS are combined, they have a total of 233 informative sites and 37 gaps (Table 4). The morphological data matrix contains 42 parsimony informative characters with 2352 data entries, of which just 214 (9.1%) were scored as unknown, 52 (2.2%) as polymorphic, and 54 (2.2%) as inapplicable. The combined data matrix including ITS, ETS, trnH–psbA, and trnS–G sequences, along with coded gaps, and morphology, has a total of 2793 characters. Among the cloned samples (Pycnandra chartacea, P. ‘‘Swenson597”, and ‘‘Sapotaceae Munzinger2786”), only those for P. chartacea yielded different ETS sequences. Variation was, however, limited to just four sites (201, A/G; 203, A/T; 310, A/G; 350, A/C). The single position (310) proved to be parsimony informative but did not influence the tree topology. The possibility of hybrid origin of any of the taxa represented by these samples was therefore rejected. 3.2. Molecular tree Both Bayesian and parsimony jackknife analyses of the combined molecular dataset recovered similar tree topologies, although the former is better-resolved (Fig. 3). A sister relationship between the Australian Niemeyera (Clade A1) and taxa from New Caledonia (Clade A2), which comprise the rest of the complex, is supported with maximum values (PP 1.00; JK 100). Several strongly supported clades are recovered but support for relationships amongst these clades are sometimes weak or absent. The tree is well resolved overall, although support for a few internal relationships is weak or nearly absent. Within the ingroup (Clade

Table 4 Characteristics of sequence and morphological characters in each of the data partitions in the phylogenetic study of the Niemeyera complex (Sapotaceae). Data

ITS ETS trnH–psbA trnS–G Morphology Total

Number of characters Aligned

Informative

Constant

Uninformative

Gaps

888 441 597 771 42 2739

130 103 11 4 42 290

648 264 560 744 0 2216

110 75 26 23 0 234

25 12 12 5 — 54

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Fig. 3. Bayesian majority-rule consensus tree (shown as a phylogram) of the Niemeyera complex (Sapotaceae) with likelihood estimated branch lengths based on combined nrDNA and cpDNA sequence data. Along the branches are Bayesian posterior probabilities (above) and parsimony jackknife support (below). Scale bar indicates the number of mutations per site. Clades A–H are discussed in the text. Generi-types are shown in bold.

A2), many clades (labeled B–G in Fig. 3) can be recognized that are well-supported by both Bayesian and jackknife analysis. Another clade (labeled H) received moderate to weak support in the molec-

ular tree, but was better supported in the combined analysis that included data from morphology (see below). Some of these clades correspond to genera recognized by Aubréville (1967), including

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‘‘Corbassona” (part of Clade D), Sebertia (Clade F), and Leptostylis (Clade G). On the other hand, our results indicate that both Pycnandra and Ochrothallus are polyphyletic. Species currently assigned to Pycnandra are distributed among no less than four clades (Clade C– E, H). Constraining the tree so that all samples of Pycnandra form a clade requires at least 40 extra steps. Similarly, terminals referred to Ochrothallus are scattered throughout the tree and forcing their monophyly would requier 34 extra steps. As reported earlier (Swenson et al., 2007a), the polyphyly of Niemeyera sensu Pennington (1991) is confirmed and constraining the tree to render samples currently assigned to the genus monophyletic would require 67 additional steps. The three accessions of Pycnandra fastuosa included in our sample are placed in a small, strongly supported clade that also includes accessions of one other member of the genus and Ochrothallus multipetalus (lower part of Clade D). However, in the Bayesian analysis only two of the three accessions form a clade, and the third one is left in an unresolved position. Pycnandra elegans and P. gatopensis, which were included by Aubréville (1967) in P. decandra (unsampled), are sister to one another within Clade H. 3.3. Combined tree (molecular + morphological data) Bayesian and parsimony jackknife analyses using the expanded dataset that also includes 42 morphological characters recovered trees with topologies similar to those obtained from molecular data alone (Fig. 4). However, the three main clades (Clades B + C + D, E, and F + G + H) collapse into a trichotomy in the Bayesian analysis, whereas the jackknife analysis provides weak support (JK 59) for a sister relationship between Clade E and F + G + H and Clades C and D form a polytomy (not shown in Fig. 4). No other conflict was found between the results of the two analyses. The strong sister group relationship between the Australian (Clade A1) and New Caledonian taxa (Clade A2) is once again found. Similarly, all but one of the clades identified among the taxa from New Caledonia using sequence data alone are recovered. The only exception is the sister relationship between Clades B + C and D, which was strongly supported in the molecular tree but is resolved as a poorly supported grade in the combined analysis. Elsewhere within Clade D, the same well-supported groups are recovered. Also, the combined tree provides strong support for an identical Clade E that comprises taxa currently assigned to three of Aubréville’s genera (1 sp. of Niemeyera, 2 spp. of Ochrothallus, and the type species of Pycnandra) along with five undescribed species. Clade F is formed by two closely related species that were placed in Sebertia by Aubréville (1967) but were transferred into Niemeyera by Pennington (1991). Our results indicate that these two taxa are sister to a clade comprising Leptostylis (Clade G) and a majority of the species currently assigned to Pycnandra (Clade H), a relationship that was indicated in previous studies (Bartish et al., 2005; Swenson et al., 2007a). In fact, the inclusion of morphology in the analyses introduces phylogenetic structure to this part of the tree, whereas the opposite is true among Clades B–D. Clade G, which exclusively includes all the species of Leptostylis, is the only group identified in our analyses whose composition fully matches the concepts of Aubréville and Pennington. In addition to the maximum levels of support found from the Bayesian and parsimony jackknife analyses, Leptostylis likewise has a Bremer support value of 22 steps based on the molecular data alone and 25 steps using the combined dataset. The combined analysis provides additional support for Clade H and for its sister relationship to Clade G. Members of Clade H are fairly homogenous morphologically, with the notable exception of Ochrothallus litseiflorus, which is the only taxon with a single stamen (rather than two) inserted opposite each corolla lobe. It also includes the generi-type of Achradotypus (Baillon, 1890), represented by Pycnandra vieillardii.

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The three accessions of Pycnandra fastuosa form a clade in the combined analysis with strong to moderate support (PP 95; JK 83) for their monophyly, and the same holds true for the clade comprising P. elegans and P. gatopensis. 3.4. Mapping of morphological characters Figs. 5 and 6 show the twelve morphological characters selected for their potential diagnostic value mapped onto the Bayesian majority-rule consensus tree generated from the molecular dataset. 4. Discussion 4.1. Tree topology Our phylogenetic analyses based on data from nuclear and chloroplast sequences combined with morphology have yielded significantly improved resolution in the Niemeyera complex compared to previous studies (Bartish et al., 2005; Swenson et al., 2007a). The vexing polytomy in earlier analyses has to a large extent now been resolved, although support for several internal relationships remains weak or moderate (cf. Figs. 3 and 4). All of the species sampled fall clearly into one of the seven clades found in both our molecular and combined trees (Clades B–H). This has important implications for the proposed re-circumscription of genera within the complex (see below), making it possible to describe twenty new species and assess their conservation status. The trees recovered from Bayesian and parsimony jackknife analyses were largely congruent, although the former provided support (albeit weak to moderate) for some relationships that were not recovered in the latter. Nevertheless, all of our analyses reveal a strongly supported clade comprising the New Caledonian members of the Niemeyera complex (Clade A2). Furthermore, within this large clade, regardless of the analysis or the dataset, seven clades (B–H) are consistently recovered. While our results lend support for some of Aubréville’s (1967) concepts of genera within the Niemeyera complex, they show that those of Pennington (1991) are untenable (Fig. 4). Niemeyera, Ochrothallus, and Pycnandra are polyphyletic, as currently circumscribed, with species of each occurring in two or more clades. 4.2. Mapping of morphology Morphological characters are known to be highly homoplasious in Sapotaceae, a situation that is evident when they are optimized on internal branches of phylogenies based on molecular data (Swenson and Anderberg, 2005; Swenson et al., 2008). Despite the difficulties this presents, the inclusion of morphological features in phylogenetic analyses can nevertheless be helpful for improving resolution in more inclusive groups (Swenson et al., 2007a). In our study of the Niemeyera complex, the value of morphological data is somewhat ambiguous. The analyses based on molecular sequences alone yield strong support for several of the groups recovered in our trees (Clades A–C and E–G), and this remains unchanged when morphology is included. Bayesian analysis of the molecular dataset likewise lends strong support for the sister relationship between Clade B + C and Clade D (PP 1.00) as well to Clade D itself (PP 1.00; Fig. 3), but in the combined analyses incorporating morphology, inferences of the relationships involving these groups are different as are their support values (Fig. 4), suggesting that the morphological dataset introduces significant phylogenetic noise. In contrast, support for Clade H is weak in the molecular phylogeny (PP 0.89; JK 56) but considerably stronger when morphology is included (PP 0.99; JK 91). This situation of discrepancies involving the positions and support of Clades B–D has

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Fig. 4. Bayesian majority-rule consensus tree of the Niemeyera complex (Sapotaceae), based on total evidence of molecular and morphological characters, with support indicated along the branches: Bayesian posterior probabilities above and parsimony jackknife below. Clades A–H are discussed in the text. Generi-types are shown in bold. Names of taxa follow Aubréville’s (1967) classification. Pennington’s (1991) generic concepts are shown to the left.

important implications for our choice of which tree to select for mapping selected morphological characters. Scotland et al. (2003) and Olmstead and Scotland (2005) argue for a limited use

of morphology and take the view that morphological characters should simply be mapped and studied on a molecular phylogeny. An opposing position is taken by Wiens (2004), who argues that

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Fig. 5. Six morphological characters mapped onto the Bayesian phylogeny of the Niemeyera complex (Sapotaceae) obtained from molecular data (Fig. 3). Letters (A–H) refer to clades in Figs. 3 and 4 as discussed in the text. Equivocal states are shown in black (j). Characters and character codings are found in Appendix A and B. (A) Leaf indumentum (char. 3), (B) tertiary venation (char. 10), (C) bracts along the pedicel (char. 12), (D) length ratio between corolla tube and lobes (char. 15), (E) corolla indument (char. 16), (F) corolla lobe orientation (char. 19).

morphology should be included in the process of constructing hypotheses of phylogeny. Our results do not offer overwhelming evidence in support of either of these alternative opinions. The fact

that both our molecular and combined analyses recovered a set of well-supported and congruent clades would tend to favor the topology derived from the combined data, which is also the most

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Fig. 6. As in Fig. 5, but six additional morphological characters. (A) Number of sepals versus petals (char. 13), (B) number of stamens inserted opposite each corolla lobe (char. 20), (C) indument on stamen filament (char. 26), (D) indument on ovary (char. 29), (E) fruit form (char. 34), (F) seed scar width (char. 38).

relevant to the analysis of morphological characters (Wenzel, 1997). On the other hand, morphology introduces noise that impacts a limited part of our phylogeny, whereas the use of a tree derived from the molecular dataset alone provides us with the opportunity to identify equivocal morphological characters. More-

over, the molecular phylogeny is unbiased with respect to morphology. Since one of our goals is to identify and analyze both problematic morphological characters and those that may be of diagnostic value for developing an improved generic level classification, we have therefore chosen to map the selected traits on the

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topology obtained exclusively from the molecular dataset (Figs. 5 and 6). 4.2.1. Hairs, leaf venation, and pedicel Presence of malpighiaceous hairs (trichomes) is a diagnostic character for Sapotaceae, and within the order (Ericales) this type of hair is only present in one other family, Ebenaceae (Metcalfe and Chalk, 1979; Kubitzki, 2004). Interestingly, a recent molecular phylogenetic study based on 11 markers from three genomes has found strong support for a close relationship between these two families (Schönenberger et al., 2005). The majority of species in Sapotaceae have indument on at least some structures. Hairs are frequently present on leaves, although many species are glabrate. Within the family, presence of indument is of little taxonomic value (Swenson and Anderberg, 2005), although details of the morphology of malpighiaceous hair have yet to be investigated across the family. For instance, taxa can be found with typical malpighiaceous hairs or with hairs that are Y-shaped, simple, or a mixture of the three types (Pennington, 2004), but nothing is known about the developmental relationship between these types or about their potential taxonomic significance. Among the taxa examined in the present study, members of the ingroup frequently have the typical form of hairs in which the two arms are round, but Y-shaped and simple hairs are also present (especially in members of Clade E). Some hairs are so tightly adpressed to the lower leaf surface that a pellicle is formed (char. 4:1). This type of indument is diagnostic for two subclades, Ochrothallus sarlinii + O. ‘‘Munzinger696” in Clade B and Niemeyera balansae—‘‘Sapotaceae Swenson615” in Clade E. The nature of the cell wall of malpighiaceous hairs, when observed under a microscope, is either distinct or indistinct (char. 5). In Clade F (Sebertia), hairs combine these character states so that each hair has a cell wall of the lower side that is distinct but the cell wall of the upper side is indistinct. The results of our analyses suggest that the presence of leaf indument in the Niemeyera complex is a plesiomorphy and that this feature has been lost at least five times with two reversals (Fig. 5A). One of the losses occurred in the ancestor to Clade G + H, an event that yields morphological support to this clade. Leaf venation has been regarded as a source of useful characters for recognizing taxa at the generic, sectional, and species level in Sapotaceae (Pennington, 1990, 1991). Variation in secondary venation is of limited value across the family (Swenson and Anderberg, 2005) and within subfamily Chrysophylloideae (Swenson et al., in press), but higher-order venation may be useful at the generic level, as exemplified by the presence of a fine areolate venation in members of Beccariella and Pichonia (Swenson et al., 2007a; Triono et al., 2007). In the group under study here, tertiary veins are horizontal (at right angles to the midrib and anastomosing with the next secondary below) in Niemeyera s.str. (i.e., the Australian species comprising Clade A1), oblique in members of Clade E, and reticulate with some veins running parallel to the secondaries near the midrib in Clade H (Fig. 5B). Overall, however, this character exhibits substantial homoplasy with numerous reversals in Clades B–D. Small bracts at the base of the pedicel subtend most sapotaceous flowers. Within the study group, some taxa of Clade C and all of Clade H have some (often 1–3) bracts distributed along the pedicel (Fig. 5C) and the uppermost bract is often sepal-like. Also, numerous imbricate bracts are present in Ochrothallus ‘‘Munzinger696”, O. sarlinii, and ‘‘Sapotaceae Munzinger2622”, three taxa with sessile flowers. 4.2.2. Corolla The corolla of Sapotaceae is partly sympetalous, comprising a tube and free corolla lobes. The fused portion varies in length and when combined with the orientation of the corolla lobes,

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which can be erect, spreading, or revolute, this influences corolla shape. Moreover, the shape and orientation of the various parts of the corolla determine the degree to which the stamens and style are exposed to pollinators, and it has been suggested that the high degree of homoplasy observed in corolla morphology throughout Sapotaceae is a response to evolutionary pressures from pollinators (Swenson et al., 2007a, 2008). The diagnostic value of corolla characters may therefore be limited to closely related genera and/or species. Our analysis shows that a corolla in which the tube and lobes are more or less equal in length is plesiomorphic for the Niemeyera complex (Fig. 5D). In ‘‘Corbassona” (part of Clade D), the tube is shorter than the lobes, although floral morphology is poorly known in the two undescribed species that belong to this group. A similar situation has evolved in the ancestor of Clade G+H, with several subsequent reversals. Within Clade G (Leptostylis), whose branch length is one of the two longest in our study, the corolla tube varies greatly in length, ranging from shorter than the lobes to longer than them. Leptostylis filipes, which occupies a basally branching position within the genus, has rather large (10–18 mm long), red, pendulous flowers with a particularly long corolla tube, whereas the more derived species L. petiolata has small (3–4 mm long), white, almost erect flowers with a short corolla tube. An intermediate floral morphology can be seen in L. goroensis (Fig. 1I). The pronounced variability in corolla features among species of Leptostylis indicates that this lineage may have been subjected to particularly strong evolutionary pressure from a diversity of pollinators and suggests that detailed studies of the pollination biology of this genus may be of interest. In general, however, the relative length of the corolla tube and lobes within a species is probably an adaptation to its specific pollinators and its diagnostic value is likely limited to separating closely related species. Within the Niemeyera complex, the corolla can either be glabrous, sparsely pubescent (often in the middle of the corolla lobes), or densely so. Our analyses indicate that the presence of corolla indument is a derived feature within the group, and that it has evolved at least twice, in Clade B + C + D and in Clade H (Fig. 5E). Within Clade B–D, while most members have a more or less pubescent corolla, this feature has been lost in some taxa, which accounts for some of the noise in our analyses. In Clade H, members of the basally branching subclade (comprising Ochrothallus litseiflorus, Pycnandra ‘‘Munzinger2615”, and P. ‘‘Munzinger2885”) have a glabrous corolla, but all other taxa in the group have a sparsely pubescent corolla (although some individual flowers may be almost glabrous), a feature that provides phylogenetic support for this part of the tree. Most Sapotaceae have spreading corolla lobes (Pennington, 1991), and it was recently suggested that the presence of revolute lobes (cf. Fig. 1B) might be a diagnostic character for the Niemeyera complex (Swenson et al., 2007a). Our results here show that while this feature represents a synapomorphy for the entire complex, reversal to spreading corolla lobes has occurred three times (Fig. 5F), once in the common ancestor to Clade H, where it represents a synapomorphy for the entire group, and a second time in Clades B + C + D, where substantial homoplasy is seen. The third case is found within Clade E, where all taxa have revolute corolla lobes except Pycnandra benthamii, the type of the genus, whose lobes are spreading and whose flowers, apart from their red color, resemble those of species belonging to Clade H (cf. Figs. 1F and G). 4.2.3. Floral anisomery and stamens In the present study we found no evidence of hybridization or concerted evolution of the kind reported by Smedmark and Anderberg (2007) in another sapotaceous genus, Nesoluma (= Sideroxylon), to explain the pattern of anisomerous flowers, in which the corolla lobes are more numerous than the five sepals. Species

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currently assigned to Ochrothallus, a genus defined in part on the presence of anisomery, are scattered among all of the clades identified by our analyses except Clade F (Sebertia) and Clade G (Leptostylis), both of which comprise exclusively anisomerous taxa (Fig. 6A). In addition, in Clade E and Clade H we find members of Pycnandra with anisomerous flowers. Moreover, the combined distribution of anisomery and the number of stamens shows an interesting pattern. None of the species with anisomerous flowers has two stamens inserted opposite each corolla lobe except for four stem taxa in Clade H (Pycnandra ‘‘Munzinger3135”, P. controversa, P. comptonii and P. ‘‘Swenson597”). In other words, with the exception of these four species, all members of the Niemeyera complex with two stamens inserted opposite each corolla lobe have isomerous flowers (Fig. 6B). Pycnandra benthamii, which has anisomerous flowers in combination with 3 or 4 stamens inserted opposite each corolla lobe, thus stands out in the complex. These findings suggest that the development of an increased number of petals and a doubling of the number of stamens are usually mutually exclusive character states, and in any case, our results show that these features are of limited diagnostic value. The presence of malpighiaceous hairs on the filament (Fig. 6C) and on the anther (char. 27) appears to have some potential diagnostic value. The distribution of these two characters seems to be correlated among members of the study group except that the taxa in Clade C have pubescent filaments and anthers, but in three members of Clade E (Niemeyera balansae, ‘‘Sapotaceae Munzinger2786”, and ‘‘Sapotaceae Swenson615”) only the anthers are pubescent. 4.2.4. Ovary and fruit As indicated above, the distribution, presence or absence, and type of malpighiaceous hairs have proven to be valuable for identifying clades within the Niemeyera complex, and this applies to indument on the ovary as well. The plesiomorphic state is for ovaries to be pubescent throughout. Some species, however, can be recognized by having a ring of hairs (variously dense) at the base of the ovary, whereas other taxa have a completely glabrous ovary. The presence of indumentum at the base of the ovary appears to be restricted to Clade H, although it is not uniformly present in this group as some members have ovaries that are pubescent throughout. A completely glabrous ovary appears to be the only unambiguous synapomorphy for Clade E (Fig. 6D). Fruits of species belonging to the Niemeyera complex are to a large extent homogenous, containing a single seed at maturity with plano-convex cotyledons, an included radicle, and no endosperm (although fruits are unknown in 10 of the 46 sampled ingroup taxa). Field observations suggest that the cotyledons are uniformly red. Variation can be seen in several other fruit characters, such as its form and the width of the seed scar. The fruits of Australian Niemeyera are generally globose, whereas among New Caledonian taxa they are variable in shape in Clades B–D, mostly cylindric in Clade E, and ellipsoid in Clade G+H (Fig. 6E). The cylindric fruit of species in Clade E, combined with a glabrous ovary, is a potential diagnostic character combination, although Pyenandra. benthamii has an ellipsoid fruit similar to that of members of Clade H. Mature fruits of Ochrothallus ‘‘Munzinger1394”, O. gordoniifolius, and ‘‘Sapotaceae Munzinger2786” are still unknown, but considering their phylogenetic position within the complex, it seems likely that they are cylindric in form. Seed scar width, and in particular its ratio to seed circumference, was an important character in the classifications of Aubréville (1967) and Pennington (1991). While details of seed morphology are inadequately known in about a dozen taxa belonging to our study group, several interesting patterns can nevertheless be noted. Nearly all members of the large group comprising Clades B, C, and D have a wide seed scar, measuring at least 60%

of the total width of the seed (Fig. 6F). The only exceptions are the two species of ‘‘Corbassona”, whose scars are much narrower (20–30%), which leads us to postulate that the two closely related undescribed species would also have narrow seed scars. Although seed features are unknown in some members of Clade E, this group seems to be characterized by moderately broad scars (about 50%). Within Clade G (Leptostylis), seed scar width is about 20–30% that of the seed in the three species for which we have data, and we expect the same will be true for the other members of the genus. We refrain from attempting to evaluate the evolution of seed scar width in Clade H because this feature varies so much within the group and is unknown in many taxa. 4.3. Ancestral reconstruction Bartish et al. (2005) suggested that the ancestor of the New Caledonian Niemeyera complex probably colonized the island through a single dispersal event from Australia in the Mid to Late Oligocene, although this interpretation has recently been questioned (Ladiges and Cantrill, 2007). However, recently generated comprehensive molecular data (Swenson et al., 2008) and more sophisticated dating estimates (Bartish et al., unpublished data) provide additional confidence in this estimate. In any case, regardless of the timing of the colonization, by mapping selected morphological characters on the phylogenetic tree based on our molecular sequence data, we now have an opportunity to reconstruct the morphological features that would have characterized the hypothetical ancestor that first reached New Caledonia (Fig. 7). Based on our analyses, this plant is thought to have had pubescent leaves, a glabrous, five-merous or anisomerous flower, more or less revolute corolla lobes, a single stamen inserted opposite each corolla lobe, and an ovary with indumentum. No extant plant with this combination of characters is known to occur in New Caledonia or Australia, but the descendants of this hypothetical ancestor appear to have changed remarkably little over a considerable period of time. 4.4. Towards a natural classification Several recent phylogenetic analyses of Sapotaceae based on combined data from cpDNA and morphology (Swenson and Anderberg, 2005), nrDNA (Bartish et al., 2005), or nrDNA and morphology (Swenson et al., 2007a), have provided strong evidence that neither of the principal classification systems of the family (Aubréville, 1967; Pennington, 1991) accurately reflects relationships among members of subfamily Chrysophylloideae in Australasia. The results presented here focusing on the Niemeyera complex add further support to these conclusions. Moreover, because we were able to achieve near-comprehensive sampling, our analyses provide a basis for proposing a new generic level classification for the group. Any new system clearly must respect the principle of monophyly and be based on well-supported clades. But in order to be of real value, the groups ought to be meaningful and distinguished by clear morphological features, and nomenclatural stability should also be maintained insofar as possible (Backlund and Bremer, 1998). The simplest way to treat the Niemeyera complex would be to include all of the species from both New Caledonia and Australia in a single, broadly defined genus, for which the name Niemeyera has nomenclatural priority. If circumscribed in this way, the genus would be characterized by a combination of the following features: absence of staminodes, indistinguishable stigmatic areas, and fruits with a single seed, included radicle, plano-convex cotyledons and no endosperm. This idea was, however, rejected by Swenson et al. (2007a) because, while it would meet the criterion of monophyly, it would result in a group that is morphologically so heterogeneous as to be of essentially no practical value for identifying or

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Fig. 7. Reconstruction of a hypothetical ancestor of the Niemeyera complex (Sapotaceae) that most probably reached New Caledonia in the Mid to Late Oligocene. (A) Habit, (B) secondary and tertiary venation on the lower surface, (C) indument, (D) transection of a flower, (E) flower, (F) fruit, (G) seed (above) and cotyledons (below). Drawing: Emma Hultén.

recognizing its members. For this reason, Swenson et al. (2007a) adopted a more narrow definition of Niemeyera, restricting the name to the five species occurring in Australia (Clade A1). An alternative approach would be to place all of the New Caledonian species (i.e., all members of our Clade A2) in a single genus, which would either bear the name Leptostylis or Pycnandra, as they were published simultaneously (Bentham and Hooker, 1876) and therefore have equal nomenclatural priority. Such a solution is potentially attractive since the group has strong phylogenetic support, and the adoption of a broadly circumscribed genus in this way would not unnecessarily compromise nomenclatural stability since both Ochrothallus and Pycnandra have in any case been shown to be polyphyletic and therefore cannot be maintained as traditionally defined. Circumscribed in this way, the genus would include species with isomerous and anisomerous flowers, and with one and two or more stamens inserted opposite each corolla lobe, but because these long-used characters have now been shown to exhibit considerable homoplasy and to be of limited diagnostic va-

lue, this does not appear particularly problematical. Treating all the New Caledonian species together would, however, still result in a group with nearly intractable morphological heterogeneity. Moreover, the many well-supported clades defined in the present study would be encompassed within a single genus, obscuring the phylogenetic information they do contain, or at least relegating it to the infrageneric level. Furthermore, if the generic name Leptostylis were retained, no fewer than 27 new combinations would be required, representing 73% of the 37 described species in the group (including unsampled taxa), and if Pycnandra were used, 25 new combinations would be necessary. A third solution would be to adopt more narrow generic concepts similar to those used by Aubréville (1967), recognizing those well-supported clades identified by our analyses that can readily be identified based on morphology. This approach would work well for Clades E through H (see below), but the delimitation of genera within the group comprising Clades B–D would be more problematical (Table 2).

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4.4.1. Clades B–D Three options are possible regarding the classification of Clades B–D: (1) treat Clades B + C + D as a single genus, (2) regard each of these clades as a separate genus, or (3) treat each well-supported subclade as a genus, including several entities within Clade D. Again, each of these would maintain monophyly, but none of them is ideal. Nevertheless, the first alternative, to amalgamate Clades B + C + D into a single genus, would yet again form a morphologically heterogeneous group, including much of the variation within Clade A2 as a whole. While Bayesian analysis using just the molecular data (Fig. 3) yields strong support (PP 1.00), jackknife analysis does not (JK 59), and when data from morphology are added, Bayesian support decreases dramatically (PP 0.65). These differences may result from a poor understanding or a lack of definition of some morphological characters, but more likely they reflect a high level of homoplasy within Clades B–D. Nevertheless, most members of a genus defined in this way would share a combination of characters including alternate leaves, presence of indument on both their leaves and ovaries, and broad seed scars (P60% of the width of the seed) (Figs. 5A, Fig. 6D and F). Such a circumscription would also include all species with a pubescent corolla and a single stamen inserted opposite each corolla lobe. Among the available generic names, Ochrothallus has priority; ‘‘Corbassona” and Trouettia would be placed in synonymy, and five new combinations would be necessary. The second alternative would recognize three genera corresponding to Clades B, C and D. The first of these, Clade B (PP 1.00; JK 85–90), which includes three entities and would require a new generic name, is characterized by (sub)sessile flowers and the presence of closely imbricate bracts that hide the very short pedicel, as well as a combination of several additional features including a glabrous corolla, a single stamen inserted opposite each corolla lobe, a conic ovary with an indistinguishable style, an ellipsoid fruit, and indistinct cell walls in the malpighiaceous hairs (Figs. 5C and E, 6B and E). However, Trouettia lissophylla varies in several of these features. Members of Clade C (PP 1.00; JK 100) would likewise represent a new genus comprising the taxa currently referred to as Ochrothallus schmidii, Pycnandra ‘‘Munzinger2624” and ‘‘Sapotaceae Munzinger2622”. The first two have five-merous flowers with two stamens inserted opposite each corolla lobe whereas the latter has anisomerous flowers with a single stamen per corolla lobe. Diagnostic features for this group include the presence of indument (often dense) on the outside of the corolla and on the filaments and anthers (Figs. 5E, 6C). Clade D (PP 6 0.65; JK 6 81) comprises three well-supported subclades (PP 1.00; JK 97–100) representing a total of nine species in our sample. This group includes the types of two genera recognized in Aubréville’s classification, or three if we include Aubréville’s superfluous name ‘‘Corbassona”, i.e., Ochrothallus sessilifolius, T. leptoclada, and C. deplanchei (the latter two are conspecific), of which the first has priority. Members of this group have five sepals, pedicels subtended at their base by small bracts, and ovaries with indument throughout, and they can be distinguished from the taxa in Clade F based on a broad seed scar and the nature of the cell wall of the malpighiaceous hairs (Figs. 5C, 6D and F). Recognizing clades B, C and D each as a separate genus would require seven new combinations. Alternative three differs from the previous one by assigning generic status to each of the three subclades within Clade D. Trouettia (= ‘‘Corbassona”) would apply to four species, two of which were recognized by Aubréville (1967) and two that are as yet undescribed. Based on currently available material, distinctive characters of this group appear to include parallel and reticulate tertiary venation, five-merous flowers with a relatively small corolla (3–5 mm long) with indument and a short tube, and seeds with a narrow scar (Figs. 5B, D, E and 6A, B, F), although floral structure is

poorly known in the two undescribed taxa. The second subclade within Clade D is represented by two Ochrothallus species, including the generi-type. None of the twelve characters mapped in our study is diagnostic for this group, but its members can easily be recognized by their more or less tomentose leaves, truncate leaf base, and the presence of indument on the margin of the corolla lobes, which are revolute (Fig. 5A and F). The third subclade would represent another new genus distinguished by a glabrous corolla and stamens and by ovoid fruits (5E, 6C, 6E). Adopting alternative three would require three new combinations. 4.4.2. Clades E–H Our analyses provide a more unambiguous generic framework for Clades E–H (Table 2). Clade E is consistently recovered in all our analyses with strong Bayesian support (PP 1.00) and moderate jackknife support (JK 81–88). It comprises nine taxa of which five are undescribed. Niemeyera balansae is clearly a member of this group, as is Pycnandra benthamii (the generi-type), traditionally considered congeneric with about 10 other species by most authors (Vink, 1957; Aubréville, 1967; Pennington, 1991; Govaerts et al., 2001). Guillaumin (1948), however, treated Pycnandra as a monotypic genus based on the presence of 5 or 6 stamens inserted opposite each corolla lobe, but this is obviously an error since there are never more than 3 or 4 stamens per corolla lobe. The mapping of selected morphological characters on our molecular tree shows parallel evolution in several features that are shared between P. benthamii and members of Clade H (especially P. comptonii). Nonetheless, Clade E is supported by a single morphological synapomorphy, a glabrous ovary (Fig. 6D), and the group can be further diagnosed by a combination of characters including oblique tertiary leaf venation, a cylindrical fruit, and a seed scar that is ±50% the width of the seed, although some character states are still poorly known in a few taxa (Figs. 5B, and 6E–F). Clade E thus corresponds to Pycnandra, and three new combinations would be required to accommodate its members. Clade F is a small group of two species, both of which were assigned to the genus Sebertia, with S. acuminata being the generi-type (Aubréville, 1967). Our results support earlier findings that these taxa are sister to one another and that they are closely related to Leptostylis (Bartish et al., 2005; Swenson et al., 2007a). Indeed, Sebertia is difficult to distinguish from members in Clades B–D based on macro-morphology alone, although the presence of leaf indumentum with indistinct upper cell walls and a distinct basal portion appears to be diagnostic. Aubréville (1967) indicated that Sebertia was unique in having its stamens inserted within the corolla tube, but our observations clearly show that they are in fact inserted in the tube orifice as in most New Caledonian members of the Niemeyera complex. The two species of Sebertia can, however, be recognized by a combination of small flowers (2–3 mm long), included stamens, and very short styles, the latter resulting in fruits that lack the persistent 1–5 mm stylar remnant typically found in other New Caledonian members of the complex. Sebertia acuminata is noteworthy for its capacity to hyperaccumulate nickel, which results in a distinctive bluish latex (Jaffré et al., 1976), a characteristic that is not shared with the second species. Our results are consistent with earlier findings that Leptostylis (Clade G) is monophyletic and that it is sister to a group of species (Clade H) usually placed in Pycnandra (Bartish et al., 2005; Swenson et al., 2007a). Leptostylis is easily recognized by its opposite leaves, four sepals, and four-locular ovaries. The species comprising our Clade H correspond to Baillon’s (1890) genus Achradotypus (typified by A. vieillardii), where all members (except Ochrothallus litseiflorus) have two stamens inserted opposite each corolla lobe (Guillaumin, 1948). Ochrothallus litseiflorus is a notable exception, which occupies a basal position and further stands out in having anisomerous flowers and a single

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stamen per corolla lobe (Fig. 6A and B). The placement of this species may seem odd, but probe mixing or confusion of samples can be ruled out. Sequences were obtained from two different samples, one for ITS and the other for ETS, and analyses based on molecular data alone using each of these two partitions place them in the same position, as did the combined analysis, in which support was strong (PP 1.00; JK 100). All members of Clade H have a distinctive type of parallel and reticulate tertiary venation (except P. comptonii), sepal-like bracts borne along the pedicel, and spreading corolla lobes (Fig. 5B, C and F). Ochrothallus litseiflorus and two undescribed species form a well-supported subclade, which can be distinguished by being entirely glabrous except for the ovaries, which bear a ring of small hairs at the base (Fig. 5A, E and 6D). Thus, Clade H corresponds to Achradotypus and seven new combinations would be necessary to accommodate its members. 4.5. Unsampled and potentially extinct taxa We were unable to sample eight previously described taxa that almost certainly belong to the Niemeyera complex, four species of Leptostylis, Ochrothallus wagapensis, Pycnandra decandra, P. paniensis, and Trouettia heteromera. Leptostylis gatopensis, L. longiflora, L. micrantha, and L. multiflora all have opposite leaves and four sepals (Aubréville, 1967), and most probably are correctly placed in that genus. They are all rare and despite extensive fieldwork we have so far not been able to relocate them, suggesting that some or all may have gone extinct due to habitat destruction. On the other hand, two species referred to as L. ‘‘sclerophyllea” (here included) and L. ‘‘amplexicaulis” (not included) are about to be described in a forthcoming generic revision. Ochrothallus wagapensis was originally described as a species of Chrysophyllum (Guillaumin, 1944), a genus only distantly related to the Niemeyera complex (Swenson et al., 2007a, Swenson et al., 2008). The type was gathered in north-central New Caledonia, between Koné and Touho. In the same area, a single individual was found in the 1970s growing along a watercourse and re-collected three times, but has not been seen since 1979, despite extensive exploration in the area. Habitat destruction in this part of New Caledonia is extensive, especially at low elevation sites, and it is possible that O. wagapensis is now extinct. Fruits were never recorded, but based on several other morphological features, including oblique tertiary leaf venation, five-merous flowers, revolute corolla lobes, a glabrous ovary, and a moderately long style, we would predict that it belongs to Clade E, in which case it should be placed in the genus Pycnandra. Pycnandra decandra is a small tree confined to the offshore island Ile Art, north of Grande Terre, and is one of a few that were once placed in Achradotypus but later transferred to Pycnandra (Vink, 1957). Vink also described P. elegans and P. gatopensis, mentioning the similarity between the former and P. decandra, although he provided no thoughts about the affinity of P. gatopensis. Aubréville (1967) included Vink’s species within P. decandra due to the absence of diagnostic characters. Our analyses agree with Aubréville’s notion that P. elegans and P. gatopensis are closely related and difficult to distinguish from each other. Given this similarity and the fact that all three taxa have indument on the lower leaf surface, we predict that P. decandra belongs in Clade H and is closely related to the two taxa represented by the samples included in our study. Furthermore, a cursory inspection of P. decandra reveals that its slender style and pedicellate flowers differ from the conical style and subsessile flowers found in P. elegans and P. gatopensis, suggesting that it may represents a distinct species worthy of recognition in the genus Achradotypus. Pycnandra paniensis was described from moist forests of northeastern New Caledonia by Aubréville (1967), who based his description on a few sterile specimens, at least two of which clearly

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belong to other taxa. Over the last few decades, several additional specimens have been collected in flower and fruit. Pycnandra paniensis exhibits all of the characters mentioned above for Ochrothallus wagapensis, along with the presence of sepals that are spreading in habit just as the flowers are about to open, all of which suggest that P. paniensis also belongs to Clade E. Moreover, P. paniensis has a cylindrical fruit, typical of members of this group, as well as sparsely pubescent anthers, a feature shared with three species comprising one of the subclades within Clade E (viz. Niemeyera balansae, ‘‘Sapotaceae Munzinger2786”, and ‘‘Sapotaceae Swenson615”), which may well be its closest relatives. Trouettia heteromera is a shrub or a tree to 10 m, growing between 500 and 1100 m altitude on ultramafic substrates in southern New Caledonia. It was described by Vink (1958) as a species of Chrysophyllum, a genus that at the time was circumscribed to include 17 species now identified as members of the Niemeyera complex. Trouettia heteromera possesses alternate leaves, indument on the lower leaf surface and ovary, and an anisomerous flower with a single stamen inserted opposite each corolla lobe (the fruit is still poorly known). It resembles T. lissophylla in many of its features, but the leaf indument has distinct cell walls rather than indistinct as in T. lissophylla. Based on this character combination we predict that T. heteromera belongs somewhere in the Clade B + C + D.

5. Conclusions The results of our analyses show that the generic classifications of Aubréville (1967) and Pennington (1991) do not adequately reflect phylogenetic relationships within the Australasian Niemeyera complex and are in need of substantial revision. The two traditionally used characters upon which these systems heavily relied, the isomerous versus anisomerous flower and the number of stamens inserted opposite each corolla lobe, while easily observed, are homoplasious and of limited use for distinguishing monophyletic groups. Moreover, our analyses show that these characters are correlated in that species with anisomerous flowers almost never have two stamens per corolla lobe (the only exception being a few members of Clade H). Thus, either an anisomerous flower or the presence of two stamens per corolla lobe seems to have evolved within a given lineage, but rarely both characters. The high degree of homoplasy found in all of the morphological features examined in our study suggests that few character states represent exclusive synapomorphies for a given lineage, even though the clades we have identified are well-supported in the molecular analyses. As pointed out previously (Swenson et al., 2007a, 2008), many genera of Sapotaceae will need to be defined on the basis of unique character combinations, and this certainly also appears to be the case for the Niemeyera complex. One of the ultimate goals of the present study is to provide a basis for developing an improved generic level classification for the Niemeyera complex so that badly needed taxonomic readjustments can be made, about 20 new species can be described, and assessments can be made of the threat status of the more than 50 species belonging to the group. Our results have confirmed and reinforced suggestions made in previous studies for recognizing some groups as distinct genera, such as Leptostylis, Niemeyera s.str., and Sebertia. We have opted not to adopt a broad generic concept that would treat the entire Niemeyera complex or its New Caledonian members (corresponding to our Clade A2) as a single genus because it would exhibit an excessive amount of morphological heterogeneity, rendering it of little practical use. Instead, we propose to use a narrower generic concept, one that reflects evolutionary relationships within the Niemeyera complex while at the same time recognizing entities that are both well-supported in our phylogenetic analyses and that present a reasonable level of morphological

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coherence. Using this approach, species in our Clades A1, E–H can be circumscribed as distinct genera, for which names are already available, viz. Niemeyera, Pycnandra, Sebertia, Leptostylis, and Achradotypus, respectively. Generic revisions, together with conservation assessments, are now being undertaken for each of these (except Niemeyera). The delimitation of meaningful genera within the group comprising our Clades B–D is more complicated. We have presented several alternative approaches that would recognize anywhere from one to five genera, each of which would meet the criterion of monophyly, but none of which offers a clearly preferable choice at present. We have therefore opted to refrain from making recommendations at this time, and instead plan to pursue further research on this particularly complex group in an effort to identify additional morphological features that might enable us to circumscribe genera on the basis of clear and unambiguous characters.

Acknowledgments We are especially grateful to Arne Anderberg for the support he provided throughout this project. Birgitta Bremer, Mats Hjertson, Johannes Lundberg, and Mats Wedin are thanked for fruitful discussions. Two anonymous reviewers provided valuable comments on the manuscript. We are grateful to Pia Eldenäs, Bodil Cronholm, and Mattias Myrenås at the molecular laboratory (MSL), Swedish Museum of Natural History, whose assistance was indispensable. We are grateful to the Conservation authorities of the North and South Provinces of New Caledonia (DDEE & DENV), who provided collecting permits and other assistance. Emma Hultén prepared the illustrations of the hypothetical ancestor. This study is part of an ongoing research project on the phylogeny and biogeography of Sapotaceae supported by the Swedish Research Council to Ulf Swenson. Appendix A Morphological characters and character states used in the cladistic study of the Niemeyera complex (Sapotaceae) in Australasia. Characters marked with an asterisk () are chosen and mapped on the Bayesian majority-rule consensus tree based on molecular data. Morphological terminology follows Harris and Harris (1997). Leaves. and venation 1. Leaves alternate (0); opposite (1). 2. Leaves spaced along branches (0); clustered at apex (1). 3*. Malpighiaceous hairs (trichomes) on adult leaves present (0); absent (1). Malpighiaceous hairs are diagnostic for Sapotaceae. They are unicellular with two arms and a small stalk. Length of each arm and/or stalk varies as well as their form and color (char. 4–7). Hairs are frequently present on young leaves (especially on the lower surface), even on leaves that quickly become glabrous (glabrate) (Fig. 5A). 4. Malpighiaceous hairs forming an indument (0); pellicle (1). Relatively long arms versus a short stalk form a tomentulose indument (the dominant type), but if the stalk is very short, the hairs are adpressed to the lower leaf surface and form a pellicle (Pennington, 1990). Sometimes the arms and stalks are of similar length and result in ‘‘Y-shaped” hairs, here termed tomentose. Other hairs are simple (no distinct arms) and form a villous indument. The homology of these structures is unclear, because discrete states are difficult and several species have different combinations of hair types. We therefore refrain from coding Y-shaped hairs but use reductive coding for simple hairs (char. 7).

5. Cell wall of the malpighiaceous hairs thin and indistinct (0); basal side distinct, thicker than remaining walls (1); thick and distinct (2). 6. Cells of malpighiaceous hairs with content, most often forming a ferruginous indument (0), empty, forming a grayish or white indument (1). Young hairs of most species in Sapotaceae have cells with a content that may soon be lost, causing the hairs to become colorless, but some hairs are empty as young and give a grayish or silvery appearance. 7. Simple hairs absent (0); present (1). 8. Leaf venation brochidodromous (0); eucamptodromous (1); craspedodromous (2). 9. Secondary venation on the lowers surface weak (0); distinct (1); prominent (2). This character can be difficult to score, especially on dry material, and images of fresh material were helpful. For example, the taxon shown in Fig. 1E has weak secondary venation, those in Fig. 1A, H–I have distinct venation, the taxa in and 1C and G have prominent secondary leaf venation. 10*. Tertiary veins parallel and reticulate (0); reticulate (1); horizontal (2); oblique (3). The tertiary venation forms different patterns depending on whether they anastomose with the midrib, secondaries, or other tertiaries. A common pattern occurs when one or two veins run parallel to the secondaries towards the midrib, but become reticulate near the margin. Reticulate tertiaries lack the parallel ones near the midvein. Horizontal tertiaries are oriented at right angles to the midrib and anastomose with the next secondary below. Oblique tertiaries form a direct link between two adjacent secondaries (Fig. 5B). Flowers 11. Flower sessile (0); subsessile (1–2 mm) (1); pedicellate (P3 mm) (2). 12*. Pedicel subtended by bract(s) (0); bracts also along the pedicel (1) (Fig. 5C). 13. Flowers isomerous (0); anisomerous (1). The number of sepals and petals is normally equal (isomerous) in Sapotaceae but they frequently differ in members of the Niemeyera complex. For example, a flower may have five sepals and eight petals, a character state termed anisomerous (Fig. 6A). 14. Sepals 4 (0); 5 (1); 6 (2). 15*. Corolla tube shorter than lobes (0); tube and lobes more or less equal (1); tube longer than lobes (2). Swenson et al. (2007a) found that the proportion of the fused part of the corolla varies in length in the Niemeyera complex and possibly conveys a phylogenetic signal. The ratio of corolla tube versus corolla lobes length was measured in millimeters under a stereomicroscope and scored for the character states (Fig. 5D). 16*. Corolla glabrous (0); hairs present (1). Some species have hairs on the corolla, frequently on the outside of the corolla lobe, while others have a few hairs in the corolla throat. These different states have not been scored because such coding would lose potential synapomorphies (Fig. 5E). 17. Corolla margin glabrous (0); hairy (1). 18. Corolla white (0); cream (1); pink or red (2); greenish (3). 19*. Corolla lobes erect (0); spreading (1); revolute (2). Revolute corolla lobes was identified as a derived character state in the studied group and possibly a diagnostic character (Swenson et al., 2007a), explaining why it was selected and mapped (Fig. 5F). Androecium 20*. Stamens inserted opposite each corolla lobe one (0); two (1); three–four (2) (Fig. 6B). 21. Stamens fixed in the tube orifice (0); below the tube orifice (1); in the middle of the tube (2).

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22. Stamens shorter than corolla (included) (0); as long as the corolla (1); longer than corolla (exserted) (2). This character is measured as a length ratio between the stamen and the corolla. Character state two is valid when the stamen filament is longer than the corolla lobe. 23. Anthers antrorse (0); versatile (1); retrorse (2). 24. Anthers flexible on the connective (0); fixed (1). 25. Anther length: <1.0 mm (0); 1.1–2.0 mm (1); 2.1–3.0 mm (2); >3.1 mm (3). This character was included in order to assess whether anther size conveys any phylogenetic signal. Character states were arbitrarily set to one mm increments. 26*. Stamen filament glabrous (0); with at least some hairs (1) (Fig. 6C). 27. Anthers glabrous (0); with at least some hairs (1). 28. Staminodes present (0); absent (1). Gynoecium 29*. Ovary hairy throughout (0); hairy at base (1); glabrous (2) (Fig. 6D). 30. Ovary 4-locular (0); 5-locular (1); P6-locular (3). 31. Style shorter than the corolla (0); as long as the corolla (1); clearly exserted from the corolla (2). 32. Ovary and style forming an ovoid or conical structure, indistinct from each other (0); distinct, style slender on top of ovary (1). 33. Style with round stigmatic areas (0); simple without visible stigmatic areas (1).

Fruits. and seeds 34*. Fruits obovoid (0); ellipsoid (1); ovoid (2); globose (3); cylindric (4). The first three character states were combined into a single state by Swenson et al. (2007a), but we retained them here to investigate if a multistate character could provide a phylogenetic signal (Fig. 6E). 35. Fruits several-seeded (0); one-seeded (1). 36. Seeds laterally compressed (0); not compressed (1). 37. Seed scar length 100% (0); ±90% (1); 680% (2). 38*. Seed scar width 610% (0); 20–30% (1); ±50% (2); P60% (3). The shape of the seed scar (length versus width) has frequently been used for generic circumscription in Sapotaceae (Pennington, 1991). Across the family, however, this character becomes homoplastic and problematic (Swenson and Anderberg, 2005), although it provides a phylogenetic signal in a more inclusive sample (Swenson et al., 2007a). Seed scar length is rather constant in the sampled taxa, but width is more variable. Width seems to be a continuous character, and limits were thus set arbitrarily by an easily observed percentage (Fig. 6F). 39. Cotyledons foliaceous (0); thick and flat (1); plano-convex (2). 40. Cotyledons white (0); red (1). 41. Radicle exserted below the cotyledon commissure (0); included in the cotyledons (1). 42. Endosperm present (0); absent (1).

Appendix B Data matrix of 42 morphological characters of New Caledonian Sapotaceae species (Chrysophylloideae). Planchonella and Niemeyera (except N. balansae) were used as outgroup. ‘‘Corbassona” (see, Section 2.1) and undescribed taxa are in quotation marks. In the matrix, inapplicable states are coded with a dash (—) and polymorphic taxa with letters: a = 0/1; b = 1/2; c = 0/2; d = 0/3; e = 2/3; f = 1/3; ? = missing data. Taxon

‘‘Corbassona deplanchei” ‘‘Corbassona intermedia” Leptostylis filipes Leptostylis goroensis Leptostylis grandifolia Leptostylis petiolata Leptostylis ‘‘sclerophyllea” Niemeyera balansae Niemeyera chartacea Niemeyera prunifera Niemeyera whitei Niemeyera ‘‘Ford2429” Ochrothallus blanchonii Ochrothallus francii Ochrothallus gordoniifolius Ochrothallus litseiflorus Ochrothallus multipetalus Ochrothallus sarlinii Ochrothallus schmidii Ochrothallus sessilifolius Ochrothallus ‘‘Munzinger696” Ochrothallus ‘‘Munzinger1394” Planchonella eerwah Planchonella kaalaensis Planchonella luteocostata Planchonella obovata

Character number 0000000001 1234567890

1111111112 1234567890

2222222223 1234567890

3333333334 1234567890

44 12

0100200000 0100200000 111---0001 1100200001 111---0011 111---0001 1100200001 0101010113 0000200112 000020011e 000020011e ?????????? 010a200113 0100200111 011---0111 011---0010 0100000211 0101000113 0100200110 0100200121 0101000011 011---0011 001---0010 0000200001 0100010010 00000a0010

b001010010 1001010010 2010200220 2010100020 2010000020 b010000020 2010000?20 2001100020 000b100120 0001100120 0001100120 ?????????? 1a11100220 1a11111020 2111000020 b111000310 0011000??0 0011100020 2101110011 2a11111120 0011100020 2a11000020 200b100?00 1001200100 0001200300 b00b100d00

0010000101 00?0000101 2210200100 0210200100 0110100100 0210000100 0220100100 0220101121 0221000101 0221000101 02?1000101 ?????????? 021010a121 0210000101 0210100122 0110000111 01???00101 02?0100102 0110211101 02?0100101 0210000100 a110200122 10?0000001 1??????001 1000000001 10?00000c1

0010112121 11 001011212? 11 2111110b21 11 211??????? ?? 2111110b?1 ?1 2111110121 11 2111110121 11 0114110221 11 211e110321 11 2113110321 11 211311032? 11 ?????????? ?? 001411022? 11 1110110221 11 001?11122? 11 00111????0 11 011??????? ?? 001b110321 11 0110110321 11 1111110321 11 0011111321 11 001a110221 11 100000000? 00 1102000000 00 0100000010 00 000d001000 00 (continued on next page)

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Appendix B (continued) Taxon

Planchonella sphaerocarpa Pycnandra benthamii Pycnandra carinocostata Pycnandra chartacea Pycnandra comptonii Pycnandra controversa Pycnandra elegans Pycnandra fastuosa JM1281 Pycnandra fastuosa JM1694 Pycnandra fastuosa JM2027 Pycnandra gatopensis Pycnandra griseosepala Pycnandra kaalaensis Pycnandra neocaledonica Pycnandra vieillardii Pycnandra ‘‘Munzinger2615” Pycnandra ‘‘Munzinger2618” Pycnandra ‘‘Munzinger2624” Pycnandra ‘‘Munzinger2885” Pycnandra ‘‘Munzinger3135” Pycnandra ‘‘Swenson597” ‘‘Sapotaceae Munzinger1438” ‘‘Sapotaceae Munzinger1717” ‘‘Sapotaceae Munzinger2622” ‘‘Sapotaceae Munzinger2786” ‘‘Sapotaceae Munzinger2963” ‘‘Sapotaceae Swenson615” Sebertia acuminata Sebertia gatopensis Trouettia lissophylla

Character number 0000000001 1234567890

1111111112 1234567890

2222222223 1234567890

3333333334 1234567890

44 12

0000000113 0100201223 0100100000 0110200000 011---0113 011---0000 0100200000 010000012e 010000012e 010000012e 0100200000 011---0000 011---000a 011---0000 011---0010 011---0010 0100201223 010020012f 011---0010 011---0000 011---0000 0000201123 011---0010 0100200111 0101010111 01000a0010 0101010113 0100100001 0100100a0f 0100000000

2001200200 201b100212 2001100011 2101010011 2111010011 21110a0011 11011a0011 0001100011 0001100011 0001100011 11011a0011 21011a0011 11011a0c11 2101110011 2101110c11 2101000011 2a01100021 2101111021 2101000011 2111111011 21?1?????? 1?01100020 2a?1?10??0 0011111010 20011a0020 20?1?????? 20011a0020 001110a020 ba11101020 100a1a0010

1000000001 1210200122 0210100101 1210100111 0210300112 0220300102 0210100111 0210100101 0210100101 0210100101 0210100111 0210210111 1210100111 0210201111 0210200101 0110100111 1220100121 0210211101 0210000111 0220311102 ????????01 0210000121 ????????0? 0110111101 0220101121 ????????0? 0220201121 0110000101 0110000101 121000010a

1103002a00 001111??2? 0a12110?21 001??????? 0111110321 0a11110321 0011110121 0112111321 0112111321 0112111321 0011110121 001a110321 0011110221 0011110321 0011110321 001??????? 0014110221 0110110321 0011110221 001??????? 0011?????? 111??????? 011??????? 001??????? 111??????? ???01???21 0114110221 0112110221 0112110221 001a11a121

00 11 11 ?? 11 11 11 11 11 11 11 11 11 11 11 ?? 11 11 11 ?? ?? ?? ?? ?? ?? ?1 11 11 11 11

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