CharaCterisation of a novel fruit type found in ehretia ( ehretiaCeae , Boraginales )

Ehretia (Ehretiaceae, Boraginales) has been divided into two major clades, characterised by fruits with four endocarpids (Ehretia i, including species of Ehretia formerly belonging to Rotula) and by fruits with two endocarpids (Ehretia ii, plus E. microphylla (= Carmona retusa) with an undivided endocarp), respectively. Both molecular (its1) and morphological data support the recognition of an additional clade, Ehretia iii (E. longiflora species group). its representatives are characterised by four endocarpids (as in Ehretia i), but differ in the presence of distinct lamellae on the abaxial surface of each endocarpid (as in Bourreria, also belonging to Ehretiaceae). however, molecular data suggest a close relationship with Ehretia ii. At least three species belong to Ehretia iii: chinese E. longiflora, eastern indian E. wallichiana, and indonesian E. javanica.


INTRODUCTION
Ehretiaceae (Boraginales) are pantropical in distribution (with centres of diversity in central America, Africa, and East Asia) and comprise about 150 species.the plants are usually subshrubs, shrubs, or trees, and otherwise have the typical asterid characters such as tetracyclic, pentamerous flowers with five antesepalous stamens and bicarpellate gynoecia.Fruit morphology provides a systematically important set of characters in Ehretiaceae (Pitot, 1939a, b;Miller, 1989;Verdcourt, 1991;Gottschling & hilger, 2001).Most of the species of Ehretiaceae have drupes (otherwise dry nutlets), with either undivided, two-, or four-parted endocarps.the individual parts of the two-and four-parted endocarps are termed endocarpids and enclose two seeds or one seed each, respectively.Undivided endocarps contain four seeds.A preliminary phylogenetic analysis (combining both molecular and morphological data) of the core representatives of Ehretiaceae, Ehretia P.Br.and Bourreria P.Br., with a discussion on character evolution, is given in Gottschling & hilger (2001).
the other large taxon of Ehretiaceae, Bourreria, comprises species which are superficially similar to Ehretia, and nomenclature is difficult because early authors did not understand relationships.however, Bourreria can easily be distinguished from Ehretia by apomorphic features, namely distinct lamellae on the abaxial surface of the endocarpids (not known elsewhere in Ehretiaceae so far), each with an additional chamber enclosing the placenta (unique in Ehretiaceae: thulin, 1987;Verdcourt, 1991;Gottschling & hilger, 2001).
in this study, we describe a novel fruit type found in some species of Ehretia, namely E. longiflora champ.ex Benth.and its relatives, which are distributed in eastern Asia and Polynesia.the systematic position of this species group (here named as Ehretia iii) is inferred from molecular data of the First internal transcribed spacer (its1) as well as morphological features, which now include a comprehensive survey of all fruit types found in Ehretia.The phylogenetic analysis supports a modified scenario for character evolution in Ehretia as proposed in Gottschling & hilger (2001).
It is difficult to calculate how many, and which, species belong to the Ehretia iii clade.Intraspecific variability, especially of vegetative traits, is known to be high in Ehretiaceae (e.g., Miller, 1989).nevertheless, many Ehretia species have been solely described on the basis of weak characters such as (highly homoplastic) leaf size and shape, without consideration of the partition of the endocarp.
Based on the distinctive fruit type, which is presented in this study, we include chinese E. longiflora, east indian E. wallichiana hook.f.& thomson ex Gamble, and indonesian E. javanica Blume.Ehretia dunniana Lév., E. pingbianensis Y.L. Liu, and E. psilosiphon r.r.Mill also share this fruit type, but they are all probably best placed in synonymy of one of the species above.

MAtEriALs AnD MEthoDs
For morphological investigation, fruits of E. longiflora (table 1) were treated with 95% [v/v] sulphuric acid for 15 minutes.After removing the parenchyma, the endocarpids were cleaned in water, dried, and photographed (ctprecisa, Agfa) with a photo macroscope (Leitz).
the sequences were manually aligned based on the secondary structure of the its1 transcript (Gottschling et al., 2001) using se-Al v2.0a72 (rambaut, 2001).the complete data matrix is available in nEXUs format on request.Phylogenetic calculations were run in PAUP* 4.0b1 (swofford, 1998) on a Macintosh computer.Likelihood and parsimony trees were generated using heuristic searches.Bootstrap analyses (criterion = parsimony, with full heuristic search: PBs; criterion = distance, with neighbour-joining search and maximum likelihood setting: DBs) were estimated based on 1000 replicates (addseq = random, nreps = 10 and starting tree obtained by neighbour-joining, respectively).Likelihood settings from the best-fit model were determined using the Aic-criterion in Modeltest 3.06 (Posada & crandall, 1998

Molecular data
The aligned ITS1 data set was 282 bp in length.82 of these sites (29%) were parsimony-informative (4.3 per taxon).Figure 1 shows the best likelihood tree (-ln = 1752.3582)calculated with the best-fit model (GTR+G+I model; number of substitution types: 6; number of distinct data patterns under this model: 137) with bootstrap support values (above branches: criterion = parsimony, below branches: criterion = distance).

Morphological data
Each fruit of E. longiflora (and its allies) contains four endocarpids.On the abaxial surface, each endocarpid bears five lamellae in a somewhat irregular arrangement (Fig. 2).On the adaxial surface, the slit-like aperture of the funicular canal is situated in a median position (black arrow in Fig. 2).The embryo inside the endocarpid is straight and elongated (shallow arrow in Fig. 2, inferred).
Figure 3 to 5 show transverse median sections of the fruit (and the carpels).Within Ehretiaceae, lamellae on the abaxial surface of each endocarpid are found only in Bourreria and in the E. longiflora species group (Fig. 3, 5).One of these lamellae (II, at the carpel border) is remarkably elongated in E. longiflora.Each placenta is protected by an outer wing of each endocarpid, which is more extensive in E. longiflora than in, e.g., Ehretia I.

The molecular tree
The maximum likelihood tree computed from the molecular data (Fig. 1) yields the same major topology as in Gottschling & Hilger (2001): Ehretia s.l. and Bourreria are sistergroups with respect to outgroup representatives.Ehretia s.l.segregates into three monophyletic assemblages, Ehretia I, Ehretia II (with E. microphylla as sisterspecies), and, additionally, Ehretia III (represented here by E. longiflora and E. wallichiana).
Ehretia was historically subdivided into three groups: species with four endocarpids (represented by Ehretia I and Ehretia III, paraphyletic), species with two endocarpids (represented by Ehretia II, monophyletic), and E. microphylla with an undivided endocarp (Gürke, 1893).However, since molecular data support the hypothesis that ʻfour endocarpidsʼ is the ancestral character state in Ehretiaceae (Gottschling & Hilger, 2001) a close relationship of Ehretia I and Ehretia III cannot be inferred from this plesiomorphy.On the contrary, Ehretia III appears to be closely related to Ehretia II (plus E. microphylla) as discussed below.

Systematic position of the Ehretia III clade and character polarities
Monophyly of Ehretia iii is supported by both molecular and morphological data.the most striking apomorphy is the very extensive wing of each endocarp that protects the voluminous placenta (Fig. 3, 5).such a structure is not found elsewhere in Ehretiaceae.
the probable systematic position of the Ehretia iii clade can be inferred from morphological features, although molecular data do not provide bootstrap support for it.two embryo morphologies are found in Ehretia (Gottschling & hilger, 2001). in Ehretia i, the embryo is strongly curved, a character that is also found in many other representatives of Ehretiaceae (e.g., Bourreria, Rochefortia sw.). the straight embryos found in Ehretia ii and E. microphylla have been considered as the apomorphic condition.Analogous character polarity is found in heliotropiaceae (Diane et al., 2002).
The Ehretia III clade has straight embryos (Fig. 2), and this most striking apomorphy argues for the close relationship of Ehretia II (plus E. microphylla) and Ehretia III.This hypothesis is supported by the topology of the maximum likelihood tree, but not by bootstrap analyses.
The well-developed placentas found in Ehretia I (Pitot, 1939b;Gottschling & Hilger, 2001) as well as in other representatives of Ehretiaceae such as Bourreria and Rochefortia have been considered as homologous (and plesiomorphic) within Ehretiaceae (Fig. 3-5).On the other hand, placentas are only weakly developed in Ehretia II (even shown for the fossil E. clausentia: Gottschling et al., 2002).This has been interpreted as a derived condition in Ehretiaceae.In this regard, Ehretia III, with an extensive placenta, represents the ancestral condition (Fig. 5).
The presence of distinct lamellae on the abaxial surface of each endocarpid have been considered as an exclusive character of Bourreria.They may function as stabilisers of the vascular bundles supplying the mesocarp.However, such lamellae are here also reported for Ehretia III (Fig. 2, 3, 5).Several alternative interpretations are possible, since Ehretia III and Bourreria are only distantly related (Fig. 1): either distinct lamellae on the abaxial surface of each endocarpid developed twice independently in Ehretiaceae (i.e., convergence hypothesis), or distinct lamellae on the abaxial surface were already present in the last common ancestor (of Bourreria and Ehretia) and were independently reduced in Ehretia I and Ehretia II (plus E. microphylla; i.e., symplesiomorphy hypothesis).Finally, lamellae might have been developed in the ancestor of Ehretiaceae, disappeared then in the ancestor of Ehretia, and subsequently reappeared in Ehretia III.The available data are not adequate to resolve this question, since a comprehensive phylogenetic analysis of Ehretiaceae is still wanted.

Phylogeography
Most representatives of Ehretiaceae are distributed on remnants of Gondwana (e.g., Bourreria, Ehretia p.p., Halgania Gaudich., Tiquilia Pers.p.p.), exceptions are found in Tiquilia p.p. and Ehretia p.p. the occurrence of predominantly Laurasian taxa in the Ehretiaceae (e.g., Ehretia ii, with a disjunction between north America and Eastern Asia) has been discussed as result of individual colonisation events (Gottschling & hilger, 2001;Gottschling et al., in press).As an example, the wide distribution of E. acuminata r.Br.(Ehretia ii) from continental Asia through Australia can then be easily interpreted as a secondary invasion into Gondwanan continents (i.e., Australia; Johnston, 1951).
the distribution of the Ehretia iii species group also appears to be primarily Laurasian (Fig. 6, with E. javanica immigrated from Asia).Based on the monophyly of Ehretia ii (plus E. microphylla) and Ehretia iii as here discussed, the colonisation of Laurasia is now not assumed for Ehretia ii exclusively (Gottschling & hilger, 2001), but for the entire group.subsequently, molecular tree topology suggests Ehretia iii and E. microphylla as two relict (and partly sympatric) endemic lineages of this monophyletic group in eastern Asia and adjacent regions.
Figure 7 summarises the results of this study.Morphological apomorphies are indicated together with nodes with high bootstrap support values.

AcKnoWLEDGEMEnts
We would like to thank hen-biau King (taiwan) and the collectors and the curators of the herbaria and botanical gardens (especially from Edinburgh, Adelaide, and singapore) in the species list for providing us with plant material, Maximilian Weigend (Berlin) and two anonymous reviewers for helpful comments on the text, and Anita Biesek and carola Müller (both Berlin) for technical work.

Fig. 1 .
Fig. 1.Maximum likelihood tree of Ehretia based on its1 sequences.important taxa are indicated, branch lengths are to scale.the numbers assigned to the branches are bootstrap support values (values under 50% not shown); numbers above branches: criterion = parsimony, numbers below branches: criterion = distance.ingroup taxa with a four-parted endocarp are shown in black, and taxa showing 'syn-mericarpy' (Gottschling & hilger, 2001) are grey.