Plant Systematics and Evolution

, Volume 280, Issue 1, pp 1–13

Evidence from fruit structure supports in general the circumscription of Apiaceae subfamily Azorelloideae

Authors

    • Department of BiologyHarbin Normal University
  • B.-E. Van Wyk
    • Department of Botany and Plant BiotechnologyUniversity of Johannesburg
  • P. M. Tilney
    • Department of Botany and Plant BiotechnologyUniversity of Johannesburg
  • G. M. Plunkett
    • Department of BiologyVirginia Commonwealth University
    • New York Botanical Garden200th St. & Kazimiroff Blvd.
  • P. P. LowryII
    • Missouri Botanical Garden
    • Département Systématique et Evolution (USM 602)Muséum National d’Histoire Naturelle
Original Article

DOI: 10.1007/s00606-009-0160-1

Cite this article as:
Liu, M., Van Wyk, B., Tilney, P.M. et al. Plant Syst Evol (2009) 280: 1. doi:10.1007/s00606-009-0160-1
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Abstract

The fruit anatomy of 51 species of Apiaceae, representing all 23 genera of the traditional tribe Mulineae (now recognized as Apiaceae subfamily Azorelloideae) and their putative relatives, was studied in detail. Most genera (Asteriscium, Azorella, Bolax, Bowlesia, Dichosciadium, Dickinsia, Diplaspis, Diposis, Domeykoa, Drusa, Eremocharis, Gymnophyton, Hermas, Homalocarpus, Huanaca, Laretia, Mulinum, Oschatzia, Pozoa, Schizeilema and Spananthe) have a combination of woody endocarps with the innermost layer of fibers arranged longitudinally and fruits that are either isodiametric or dorsally compressed (never laterally compressed), with lateral ribs or wings that are usually larger than all other ribs or wings. This combination of anatomical characters is unique to most of subfamily Azorelloideae. Choritaenia, and Klotzschia, however, lack prominent lateral ribs or wings, and also differ in other anatomical features, suggesting the exclusion of these taxa from the Azorelloideae. Carpological characters were found to be helpful in refining the circumscription of the subfamily.

Keywords

ApiaceaeUmbelliferaeApialesAzorelloideaeCarpologyFruit anatomyPhylogeny

Introduction

Recent phylogenetic studies based on molecular sequence data have shown that the family Apiaceae, as traditionally circumscribed, is not a monophyletic group (Plunkett et al. 1996, 1997, 2004; Downie and Katz-Downie 1999; Downie et al. 2001; Plunkett 2001; Valiejo-Roman et al. 2002; Chandler and Plunkett 2004). In particular, many of these studies have demonstrated that Drude’s (1897–1898) subfamily Hydrocotyloideae is polyphyletic, with some members (including Trachymene Rudge and the subfamilial type Hydrocotyle L.) belonging to the closely related family Araliaceae. Other genera, including Centella L., Micropleura Lag., Actinotus Labill., Platysace Bunge, and two former araliads (Mackinlaya Hook.f. and Apiopetalum Baill.), were assigned to a newly described apiaceous subfamily, Mackinlayoideae (Plunkett et al. 2004). A second new subfamily, Azorelloideae, was described to accommodate most of the remaining hydrocotyloid genera, and is roughly equivalent to the hydrocotyloid tribe Mulineae as defined by Drude (1897–1898) and by Pimenov and Leonov (1993) (including Azorella, Bolax, Bowlesia, Dickinsia, Dichosciadium, Diplaspis, Eremocharis, Huanaca, Mulinum, Schizeilema and Spananthe; Plunkett et al. 2004). These realignments have substantially clarified relationships within Apiaceae, and provide a renewed framework for examining patterns of character evolution within the family and identifying potentially valuable diagnostic features for recircumscribed infra-familial groups.

Despite the consistent picture of relationships that has emerged in Apiaceae on the basis of molecular data (from a variety of markers and techniques), the complex patterns of morphological convergences and parallelisms in the family have made it difficult to identify structural characters that can be used to help define these clades. Fruit micromorphology and anatomy, however, have proved to be a major exception to this trend (e.g., see Liu et al. 2006). Thus, for the present paper, we investigated the fruit structure of genera previously assigned to tribe Mulineae to explore the taxonomic potential of these data to define clades and diagnose monophyletic groups. In this study, we examine fruit morphology and anatomy from all “hydrocotyloid” genera having a woody endocarp and isodiametric or dorsally compressed fruits. The results of this survey are then used to identify carpological features that support or refute the findings of molecular phylogenetic studies, and to predict the placement of several genera that were not sampled in previous DNA analyses (e.g., Plunkett 2001; Plunkett et al. 2004; Chandler and Plunkett 2004).

Materials and methods

Taxon sampling

Mature fruits of 51 species were studied in detail (Table 1), representing all 23 genera of tribe Mulineae (sensu Drude 1897–1898, Pimenov and Leonov 1993) and their putative relatives. Mackinlaya confusa, a member of Apiaceae subfamily Mackinlayoideae, was also examined for comparative analysis. Authorities for genus and species names, as well as voucher specimens, are given in Table 1 and are not repeated elsewhere.
Table 1

List of taxa from Azorelloideae (and putative relatives) sampled for fruit anatomical studies, together with voucher specimen details

Species

Voucher specimens or accession number and herbarium

Origin

Asteriscium chilense Cham. & Schltdl.

Worth & Morrison 16376 (K)

Argentina

Azorella compacta Phil.

Hill 192 (K)

Peru

A. corymbosa Pers.

Ramsay & Merrow-Smith 376 (K)

Ecuador

A. incisa Wedd.

Constance & Sparre 3564 (K)

Chile

A. monantha Clos

Chandler & Bayer 1113 (PRE)

Argentina

A. multifida Pers.

Hutchison 1625 (F)

Peru

A. trifurcata Pers.

Pederser 14438 (F)

Argentina

Bolax gummifera (Lam.) Spreng.

Dollenza 147 (GH)

Argentina

Bowlesia incana Ruiz & Pav.

Dale Thones et al. 17972 (NY)

USA

B. tenera Spreng.

Pleijer s.n. (S)

Sweden

Choritaenia capensis Burtt Davy

Hanekom 1834 (PRE)

South Africa

Dichosciadium ranunculaceum (F.Muell.) Domin

Verdon 2678 (CBG)

Australia

Dickinsia hydrocotyloides Franch.

NAS 403956 (NAS)

China

Diplaspis hydrocotyle Hook.f.

Verdon 2671 (U)

Australia

D. nivis Van den Borre & Henwood

Jan 1954 (NE)

Australia

Diposis bulbocastanum DC.

Pirion 1741 (GH)

Chile

D. saniculifolia DC.

Gibert 426 (K)

Uruguay

Domeykoa amplexicaulis (H.Wolff) Mathias & Constance

Stafford 805 (K)

Peru

D. saniculifolia Mathias & Constance

Ellenberg 2730 (U)

Chile

Drusa oppositifolia DC.

Jahandiez 37712 (RAB)

Morocco

Eremocharis fruticosa Phil.

Johnston 5247 (GH)

Peru

E. longiramea I.M.Johnst.

Hutchison & Wright 3487 (U)

Peru

E. triradiata I.M.Johnst.

Solomon 3064 (F)

Peru

Gymnophyton flexuosum Clos

Looser 4259 (GH)

Chile

G. isatidicarpum (C.Presl ex DC.) Mathias & Constance

Schlegel 5867 (F)

Chile

G. polycephalum Clos

Werdermann 154 (U)

Chile

G. robustum Clos

Zollner 5128 (U)

Chile

Hermas capitata L.

Bolus 9111 (PRE)

South Africa

H. ciliata L.f.

Pillans 6742 (BOL)

South Africa

H. villosa Thunb.

Compton 16844 (PRE)

South Africa

Homalocarpus bowlesioides Hook. & Arn.

Philipps s.n. (K)

Chile

H. dichotomus (Poepp. ex DC.) Mathias & Constance

Mantero 308 (K)

Chile

Huanaca acaulis Cav.

Donat 128 (U)

Chile

H. andina Phil.

Werdermann 1340 (U)

Chile

Klotzschia brasiliensis Cham.

Iewin et al. 21900 (SP)

Brazil

K. glaziovii Urb.

Ratter & Bridgwate 7227 (K)

Brazil

K. rhizophylla Urb.

Anolusen et al. 36097 (SP)

Brazil

Laretia acaulis Gill & Hook.

Werdermann 646 (U)

Chile

L. yareta (Hamman) Mathias & Constance in R.L.Rodrig.

Venturi B161 (K)

Chile

Mulinum axilliflorum Griseb.

Fiebrig 2601 (PRE)

Argentina

M. echinus DC.

Constance 3832 (K)

Patagonia

M. leptactanthum Phil.

Elwre s.n. (K)

Chile–Argentina border

M. ovalleanum Phil.

Constance 3532 (K)

Chile

M. spinosum Pers.

Mutchison 3051 (NY)

Chile

Oschatzia cuneifolia (F.Muell.) Drude

Briggs 4782 (NSW)

Australia

O. saxifraga (Hook.f.) Walp.

Grof 10133 (CBG)

Australia

Pozoa coriacea Lag.

Werdermann 611 (U)

Chile

P. volcanica Mathias & Constance

Constance & Sparre 3578 (K)

Chile

Schizeilema colensoi Domin

CHR 286768 (CHR)

New Zealand

S. haastii Domin

CHR 330239 (CHR)

New Zealand

Spananthe paniculata Jacq.

Sagastegui 10524 (MO)

Peru

Mackinlaya confusa Hemsl.

Plunkett, Jensen & Oskolski 1549 (VCU)

Australia

The genus Mackinlaya (Apiaceae subfamily Mackinlayoideae) was included in the study for comparison

Herbarium acronyms follow Holmgren et al. (1990)

Anatomical studies

After rehydration, fruits obtained from herbarium specimens were placed in FAA for a minimum of 24 h and then prepared for sectioning using the glycol methacrylate (GMA) method of Feder and O’Brien (1968), slightly modified by allowing a minimum of 5 days for the third infiltration. A Porter-Blüm ultramicrotome was used to prepare transverse sections ca. 5 μm thick. Samples were stained using the periodic acid-Schiff/toluidine blue method (Feder and O’Brien 1968). Drawings were made using a camera lucida. Terminology follows Kljuykov et al. (2004). The surface structures of the samples were observed.

Results

Variation in fruit structure, including fruit shape, mericarp surface, structure of the mesocarp and endocarp, fruit vasculature, secretory structures, structure of the commissure and carpophore, and type and arrangement of any crystals, was characterized and described for each of the 51 species (Figs. 126, 2752, 5364). A summary of the main characters examined is provided in Table 2.
Table 2

Summary of fruit characters of Azorelloideae (and putative relatives)

Species

Mericarps triangular

Mericarps dorsally compressed (or other)

Wing type (s) if present

Mericarp with five prominent ribs on dorsal side

Trichome type if present

Oil vesicles

Irregular vittae

Orientation of fibers (innermost layer of endocarp)

Groove in endosperm (if present)

Asteriscium chilense

+

++a

Lateral

Longitudinal

Azorella compacta

++

Marginal

Longitudinal

A. corymbosa

+b

Longitudinal

A. incisa

+

++

Longitudinal

A. monantha

+c

+

Longitudinal

A. multifida

+c

++

Longitudinal

A. trifurcata

++

Longitudinal

Bolax gummifera

+

++

Multicellular stellate

Longitudinal

Bowlesia incanad

+

+

Multicellular stellate

Longitudinal

B. tenerad

+

+

Multicellular stellate

Longitudinal

Choritaenia capensis

++

Marginal

Unicellular

+

Transverse

Dichosciadium ranunculaceum

+

++

Multicellular stellate

Longitudinal

Dickinsia hydrocotyloides

+

++

+

Longitudinal

Diplaspis hydrocotyle

+

++

Longitudinal

D. nivis

+

++

Longitudinal

Diposis bulbocastanum

+

++

Lateral

Longitudinal

D. saniculifolia

+

++

Lateral

Longitudinal

Domeykoa amplexicaulis

+

++

Longitudinal

Deep

D. saniculifolia

+

+

Longitudinal

Deep

Drusa oppositifolia

+

++

Lateral

Multicellular stellate

Longitudinal

Eremocharis fruticosa

+

+

Longitudinal

Deep

E. longiramea

+

+

Longitudinal

Deep

E. triradiata

+

+

Longitudinal

Deep

Gymnophyton flexuosum

+

++

Lateral

Longitudinal

G. isatidicarpum

+

++

Lateral

Longitudinal

Shallow

G. polycephalum

+

++

Lateral

Longitudinal

G. robustum

+

++

Lateral

Longitudinal

Shallow

Hermas capitata

+

+

+

Longitudinal

H. ciliata

+

++

Median, lateral

+

Longitudinal

H. villosa

+

++

Median, lateral

+

Longitudinal

Shallow

Homalocarpus bowlesioides

+

++

Multicellular stellate

Longitudinal

H. dichotomus

+

+

Multicellular stellate

Longitudinal

Huanaca acaulis

+

++

Longitudinal

H. andina

+

++

Longitudinal

Klotzschia brasiliensis

++

+

Multicellular stellate

+

Different directionse

K. glaziovii

++

Marginal (narrow)

+

Multicellular stellate

+

Different directions

K. rhizophylla

++

+

Multicellular stellate

+

Different directions

Laretia acaulis

++

Marginal

Longitudinal

L. yareta

+

++

Longitudinal

Mulinum axilliflorum

+

++

Lateral

Longitudinal

M. echinus

+

++

Lateral

Longitudinal

M. leptactanthum

+

++

Lateral

Longitudinal

M. ovalleanum

+

++

Lateral

Longitudinal

M. spinosum

+

++

Lateral

Longitudinal

Oschatzia cuneifolia

+

Longitudinal

O. saxifraga

Isodiametric

Longitudinal

Pozoa coriacea

+

++

Longitudinal

P. volcanica

+

++

Longitudinal

Schizeilema colensoi

+

++

Longitudinal

S. haastii

+

+

Longitudinal

Spananthe paniculata

+

++

Longitudinal

Mackinlaya confusa

Laterally compressed

Different directions

aStrongly dorsally compressed (dd): mericarp width is more than two times the mericarp thickness

bSlightly dorsally compressed (d): mericarp width is 1.3−2 times the mericarp thickness

cAlthough the triangular mericarps are not obvious in Azorella monantha and A. multifida, both taxa have three ribs on the dorsal side and two in the commissure

dAccording to Mathias and Constance (1965), Bowlesia incana and B. tenera are synonyms

eDifferent directions: fibers are arranged transversely, longitudinally and obliquely

Fruit shape

All taxa studied have dorsally compressed mericarps except Mackinlaya confusa (Fig. 1), a member of Apiaceae subfamily Mackinlayoideae, and Oschatzia saxifraga (Fig. 47). The mericarps may be strongly compressed dorsally (e.g., Asteriscium chilense and Dickinsia hydrocotyloides, Figs. 2, 16) or slightly compressed dorsally (e.g., Bowlesia incana and Oschatzia cuneifolia, Figs. 10, 46). Three groups of species can be distinguished based on the mericarp shape, the position of prominent fruit ribs/wings and features of the endocarp.
https://static-content.springer.com/image/art%3A10.1007%2Fs00606-009-0160-1/MediaObjects/606_2009_160_Fig1-26_HTML.gif
Figs. 1−26

Transverse sections of the fruits of Mackinlaya confusa (Apiaceae subfamily Mackinlayoideae) and members of subfamily Azorelloideae showing variation in mericarp shape, trichomes, wing types, secretory oil ducts, lignification of the mesocarp and endocarp, commissural width and features of the carpophore. 1 Mackinlayaconfusa. 2 Asteriscium chilense. 3 Azorella trifurcata. 4 Azorellamonantha. 5 Azorella compacta. 6 Azorella incisa. 7Azorella multifida. 8Azorella corymbosa. 9Bolax gummifera. 10Bowlesia incana. 11 Bowlesia tenera. 12 Choritaenia capensis. 13 Domeykoa amplexicaulis. 14 Domeykoa saniculifolia. 15 Drusa oppositifolia. 16Dickinsia hydrocotyloides. 17 Dichosciadium ranunculaceum. 18Diplaspis hydrocotyle. 19 Diplaspis nivis. 20 Diposis saniculifolia.21 Diposis bulbocastanum. 22 Eremocharis fruticosa. 23 Eremocharis triradiata. 24 Eremocharis longiramea. 25 Gymnophyton polycephalum. 26 Gymnophyton robustum. open circles secretory oil ducts and vesicle; filled circles vascular bundles and carpophore; hatching lignification; ac air chamber; b bristle; cp carpophore; e endocarp; lr lateral rib; m mesocarp; lr (lw) lateral rib (lateral wing); mar marginal rib; mar (maw) marginal rib (marginal wing); mer median rib, rd rib duct, v vesicle; vb vascular bundle; vvb ventral vascular bundle. Scale bar 0.7 mm in Fig. 1 and 1 mm in Figs. 226

https://static-content.springer.com/image/art%3A10.1007%2Fs00606-009-0160-1/MediaObjects/606_2009_160_Fig27-52_HTML.gif
Figs. 27−52

Transverse sections of the fruits of members of Apiaceae subfamily Azorelloideae showing variation in mericarp shape, trichomes, wing type, secretory ducts, lignification of the endocarp, commissural width and features of the carpophore. 27 Gymnophyton flexuosus. 28 Gymnophyton isatidicarpum. 29 Homalocarpus bowlesioides. 30 Homalocarpus dichotomus. 31Hermas villosa. 32Hermas capitata. 33 Hermas ciliata. 34 Huanaca acaulis. 35 Huanaca andina. 36 Klotzschia brasiliensis. 37 Klotzschia glaziovii. 38 Klotzschia rhizophylla. 39 Laretia acaulis. 40 Laretia yareta. 41 Mulinum axilliflorum. 42 Mulinum echinus. 43 Mulinum spinosum. 44 Mulinum leptactanthum.45 Mulinum ovalleanum. 46 Oschatzia cuneifolia. 47 Oschatzia saxifraga. 48 Pozoa coriacea. 49 Pozoa volcanica. 50 Schizeilema haastii. 51 Schizeilema colensoi. 52 Spananthe paniculata. open circles secretory oil ducts; filled circles vascular bundles and carpophore; hatching lignification; iv irregular vittae; co commissure; mar (maw) marginal rib (marginal wing); mer (mew) median rib (median wing). Scale bar 1 mm

https://static-content.springer.com/image/art%3A10.1007%2Fs00606-009-0160-1/MediaObjects/606_2009_160_Fig53-64_HTML.jpg
Figs. 53−64

Multicellular stellate trichomes (53−56) occurring on the fruit stalk and fruit epidermal surfaces. 53Dichosciadiumranunculaceum. 54Klotzschia glaziovii. 55 and 56Drusa oppositifolia. 57 Irregular vittae in Hermas ciliata. Transverse sections (58−64) showing multicellular stellate trichome, unicellular trichomes, rib duct, vascular bundles, woody mesocarp, woody endocarp, carpophore and crystals. 58 Domeykoa saniculifolia. 59 Homalocarpus dichotomus. 60 Laretia yareta. 61 Gymnophyton robustum. 62 Azorella monantha. 63 Choritaenia capensis. 64 Klotzschia brasiliensis. cp carpophore; cr crystal; e non-lignified cell of endocarp; m mesocarp; rd rib duct; t testa; ut unicellular trichome; vb vascular bundle. Scale bar 0.1 mm

Group 1. Mericarps usually triangular, lateral ribs nearly always prominent or winged and endocarp with the innermost layer of fibers longitudinally arranged—The following taxa have mericarps which are more or less triangular and which have three dorsal ribs and two commissural ribs with the lateral ribs being more prominent than the others: all species of Asteriscium (Fig. 2), Azorella (Figs. 4, 6, 7) (except Az. trifurcata, Az. compacta and Az. corymbosa, whose mericarps have prominent marginal ribs or wings, Figs. 3, 5, 8), Bolax (Fig. 9), Bowlesia (Figs. 10, 11), Dickinsia (Fig. 16), Dichosciadium (Fig. 17), Diplaspis (Figs. 18, 19), Diposis (Figs. 20, 21), Domeykoa (Figs. 13, 14), Drusa (Fig. 15), Eremocharis (Figs. 2224), Gymnophyton (Fig. 2528), Hermas (Figs. 31–33), Homalocarpus (Figs. 29, 30), Huanaca (Figs. 34, 35), Laretia (Fig. 40) (except Laretia acaulis, whose mericarps have marginal wings, Fig. 39), Mulinum (Figs. 4145), Pozoa (Figs. 48, 49), Schizeilema (Figs. 50, 51) and Spananthe (Fig. 52). Lateral wings (e.g., see Figs. 2, 20), are found in Asteriscium, Drusa, Diposis, Gymnophyton, Hermas (except He. capitata, Fig. 32) and Mulinum (Figs. 2, 15, 20, 21, 2528, 31, 33, 4145 (Table 2). A basal median wing is also present in Hermas villosa and He. ciliata. The height of sectioning is critical, so that the median wing will not be visible in transverse sections (see He. ciliata, Fig. 33) if they are made above the wing). In Oschatzia the two mericarps are not triangular and are slightly dorsally compressed in O. cuneifolia (Fig. 46) and isodiametric in O. saxifraga (Fig. 47).

A woody endocarp, composed of several layers of fibers (e.g., see Figs. 58, 60, 62), which extends into the wings (e.g., see Fig. 61), is present in all of the above taxa. These layers of fibers are arranged longitudinally, transversely and obliquely, but the fibers of the innermost layer around the seed are all arranged longitudinally (e.g., Figs. 58, 62).

Group 2. Mericarps not triangular, wings marginal, endocarp with the innermost layer of fibres mainly transversely arranged—This group contains only a single species, Choritaenia capensis, and is characterized by the presence of mericarps that are markedly dorsally compressed, with apparently two marginal wings (Fig. 12) each developing from two marginal vascular bundles. However, although there are seven vascular bundles (see below) it is likely that the two marginal bundles near the base of each wing of a mericarp represent the marginal bundle that has split into two, thus resulting in a total of eight bundles in the marginal region of the entire fruit. The endocarp does not appear to extend into the wings—this is an important difference between this species and the other azorelloids. There are indications that a few of the innermost cells of the endocarp may sometimes remain non-lignified or are at least less prominently lignified than the adjoining layers (Fig. 63).

Group 3. Mericarps not triangular, ribs five and prominent, endocarp with the innermost layer of fibers arranged in different directions—This group comprises the three species of the genus Klotzschia (K. brasiliensis, K. glaziovii, and K. rhizophylla), which all have five prominent ribs per mericarp (Figs. 3638), although the two marginal ribs may be larger than the other ribs, or slightly winged (Fig. 37). The woody endocarp in these three taxa is composed of longitudinal fibers, except for the innermost layer around the seed, where the orientation is variable (transverse, longitudinal and oblique) (Fig. 64).

Mericarp surface, mesocarp

All of the species examined have mericarps with a smooth surface except Bolaxgummifera (Fig. 9), Bowlesia (Bo. incana, Bo. tenera, Figs. 10, 11), Choritaenia capensis (Fig. 12), Drusa oppositifolia (Figs. 15, 55), Homalocarpus (H. bowlesioides, H. dichotomus, Figs. 29, 30, 59), and Klotzschia (K. brasiliensis, K. glaziovii, and K. rhizophylla, Figs. 3638, 54) (Table 2). Of these ten taxa, the mericarps of Choritaenia capensis are unique in having dense, unicellular trichomes (Figs. 12, 63) that are thick on the dorsal side and thin along the margins. The nine remaining taxa have multicellular stellate trichomes (e.g., Fig. 59) that are dense in each of the two species studied of Bowlesia and Homalocarpus, and Klotzschiaglaziovii, but sparse in Drusa oppositifolia and two species of Klotzschia (K. brasiliensis and K. rhizophylla). In Drusa, multicellular stellate trichomes also occur at the tips of several peculiar, spine-like marginal appendages or bristles (Fig. 56). The multicellular stellate trichomes in Klotzschia (e.g., Fig. 54) are soft but hard in the other taxa. Multicellular stellate trichomes with long stalks were observed on the fruiting pedicel of Dichosciadium ranunculaceum (Fig. 53) but are not found on the epidermal surface of the fruit. None of the taxa studied here have a lignified mesocarp except Bowlesia incana (Fig. 10), whose entire fruit wall is lignified around a large air chamber, and Choritaenia capensis (Fig. 63), in which it appears that several layers of the mesocarp as well as at least part of the endocarp are woody. A ring of mesocarp cells containing rhomboidal crystals of calcium oxalate (e.g., see Fig. 58) is present adjacent to the endocarp (also in the wings) in all taxa studied except for Choritaenia capensis which completely lacks crystals.

Vascular bundles in mericarps

Mackinlayaconfusa (Fig. 1) has branching vascular bundles that, in transverse section, appear as roughly seven strands in each carpel. Choritaenia capensis (Figs. 12, 63) has seven bundles in each mericarp, of which five are dorsal and two are commissural. However, as mentioned previously, it is likely that the marginal bundles split during fruit development. In all the other taxa examined, each mericarp has five vascular bundles (e.g., see Fig. 3), either three dorsal and two commissural (e.g., see Fig. 2) or all five on the dorsal side (e.g., see Fig. 5). Vascular bundles are very small and comprise only a few cells (Figs. 58, 63), which are adjacent to the endocarp in Diposis bulbocastanum, Gymnophyton isatidicarpum, Homalocarpus bowlesioides, Hermas capitata (Figs. 21, 28, 29, 32), and the three species of Klotzschia (Figs. 3638).

Secretory structures

Three types of secretory structures are present in the taxa studied, namely rib secretory ducts, irregular vittae and vesicles. Rib secretory ducts are typically located to the outside of the vascular bundles in each primary rib and are present in almost all of the species examined. Three ducts are found on the dorsal side and two on the commissure (e.g., Fig. 2), or five on the dorsal side (e.g., Fig. 3). Rib secretory ducts are absent in Choritaenia capensis (Fig. 12), indistinct in Diposis bulbocastanum (Fig. 21), Diplaspis nivis (Fig. 19), Homalocarpus dichotomus (Fig. 30), Hermas capitata (Fig. 32), Klotzschia brasiliensis, and K. rhizophylla (Figs. 36, 38). By contrast, these ducts are large in Azorella trifurcata, Az. compacta (Figs. 3, 5), Gymnophyton robustum (Fig. 26), Laretia acaulis, and L. yareta (Figs. 39, 40). Some of the ribs in the fruits of Eremocharis fruticosa (Fig. 22), E. triradiata (Fig. 23), Gymnophyton robustum (Fig. 26), G. isatidicarpum (Fig. 28) and Oschatzia saxifraga (Fig. 47) may have more than one duct. Irregular (branching and anastomosing) vittae are present in Dickinsia hydrocotyloides (Fig. 16), all species of Hermas (Figs. 3133, 57), and Klotzschia (Figs. 3638). Large oil vesicles (orbicular or disc-shaped hollows) are present in the woody mesocarp of the fruit wings of Choritaenia capensis (Fig. 12). Mackinlayaconfusa does not have five rib ducts, but instead reticulate secretory ducts are observed, which are usually located to the outside of the vascular bundles (Fig. 1).

Commissure

The commissure is the area of attachment between the two mericarps (e.g., Fig. 47). Almost all of the taxa studied have a narrow commissure, varying from 2% (e.g., Fig. 2) to 20% of the total mericarp width (e.g., Fig. 5). A wide commissure (about 50% of the mericarp width) is present in Azorellamonantha (Fig. 4), and three species of Klotzschia (Figs. 3638). Two taxa, Mackinlayaconfusa (Fig. 1) and Choritaenia capensis (Fig. 12), have very wide commissures, extending 90 and 100% of the mericarp width, respectively. Conspicuous grooves in the endosperm are visible at the commissural side in all taxa of Domeykoa (Figs. 13, 14) and Eremocharis (Figs. 2224), whereas shallower grooves are found in Gymnophyton robustum, G.isatidicarpum and Hermasvillosa (Figs. 26, 28, 31).

Ventral vascular bundles and carpophore

The carpophore varies in the taxa studied. One entire carpophore is present in Asteriscium chilense (Fig. 2), Azorella compacta (Fig. 5), Bolax gummifera (Fig. 9), Bowlesia tenera (Fig. 11), Choritaenia capensis (the carpophore is very short and therefore not visible in median transverse section, Fig. 12), Dickinsia hydrocotyloides (Fig. 16), Diplaspis hydrocotyle, D. nivis (Figs. 18, 19), Drusa oppositifolia (Fig. 15), Gymnophyton polycephalum, G. flexuosus (Figs. 25, 27), and all species of Homalocarpus (Figs. 29, 30), Hermas (Figs. 3133), Huanaca (Figs. 34, 35), Klotzschia (Figs. 3638), Laretia (Figs. 39, 40), and Pozoa (Figs. 48, 49). A carpophore with two branches, which are arranged in a plane parallel to the mericarps, occurs in Gymnophyton isatidicarpum, Oschatzia cuneifolia and O. saxifraga (Figs. 28, 46, 47), whereas one carpophore with two branches arranged in a plane at right angles to the mericarps, is present in Diposis saniculifolia, D. bulbocastanum (Figs. 20, 21), Gymnophyton robustum (Fig. 26), Mulinum echinus and M. ovalleanum (Figs. 42, 45). The carpophore may be large, as in Hermas (Figs. 3133) and Oschatzia (Figs. 46, 47), small, as in Laretia yareta (Fig. 40), or absent, as in all other taxa (e.g., Fig. 3). In Mackinlaya confusa a carpophore is absent, and the two ventral bundles are surrounded by parenchymatous cells (Fig. 1).

Discussion

All genera currently placed in Apiaceae subfamily Mackinlayoideae, including those historically assigned to subfamily Hydrocotyloideae, have laterally compressed mericarps, as exemplified by Mackinlaya confusa (Fig. 1). The remaining genera traditionally placed in Hydrocotyloideae have more or less dorsally compressed mericarps, and can be divided into three groups according to mericarp shape, rib/wing configuration and endocarp structure.

Group 1. Taxa with lateral wings and the innermost layer of fibers of the woody endocarp arranged longitudinally—This group includes Asteriscium, Azorella, Bolax, Bowlesia, Dichosciadium, Dickinsia, Diplaspis, Diposis, Domeykoa, Drusa, Eremocharis, Gymnophyton, Hermas, Homalocarpus, Huanaca, Laretia, Mulinum, Oschatzia, Pozoa, Schizeilema and Spananthe. The mericarps of all these taxa usually have lateral ribs or wings that are more prominent than the other ribs or wings (e.g., Fig. 2), a feature that appears to be unique for most of the taxa studied here, including all those sampled by Chandler and Plunkett (2004) for their molecular analysis and assigned to the newly established apiaceous subfamily Azorelloideae (Plunkett et al. 2004), namely Azorella, Bolax, Bowlesia, Dichosciadium, Dickinsia, Diplaspis, Eremocharis, Gymnophyton, Huanaca, Mulinum, Schizeilema and Spananthe. Prominent lateral wings may thus represent a synapomorphy for Azorelloideae, and their presence in several genera for which molecular data are lacking (viz., Asteriscium, Diposis, Domeykoa, Drusa, Hermas, Homalocarpus, Laretia and Pozoa) suggests that the subfamily should now be expanded to include them. Although Oschatzia does not have prominent lateral ribs (or wings), it is considered to be part of this group. Future molecular studies should target these taxa to confirm their placement in Azorelloideae.

Multicellular stellate trichomes occur in Bolax (Fig. 9), Bowlesia (Figs. 10, 11), Drusa (Fig. 15), Homalocarpus (Figs. 29, 30), Klotzschia (Figs. 3638), and Dichosciadium (Fig. 53), but are not known in any other genera of Apiaceae. Mathias and Constance (1965) regarded Drusa and Homalocarpus as closely related. Hakansson (1952) confirmed the presence of bipolar embryo sacs in both Bowlesia and Drusa, and Henwood and Hart (2001) placed Drusa with Homalocarpus because they both have calyces forming an entire rim, stellate trichomes and opposite leaves; the latter two features are also present in Bowlesia. Elsewhere in the family, stellate trichomes occur only in Marlothiella gummifera H.Wolff., but these are unicellular (Liu et al. 2007b) and thus not homologous to the multicellar trichomes found in Azorelloideae. Choritaenia has many unicellular, simple trichomes (Burtt 1991), as do several other genera of Apioideae, such as Ezosciadium B.L. Burtt, Magydaris W.D.J.Koch ex DC., and Tordylium L. To date, uniseriate multicellular hairs have been found only in Cannaboides betsileensis (Humbert) B.-E. van Wyk and the forma of Heteromorpha involucrata Conrath previously known as H. kassneri H.Wolff (Liu et al. 2006).

Domeykoa and Eremocharis share several features, including a flat to concave surface on the dorsal side of the mericarp, and a deep groove on the commissural face of the endosperm. On the basis of morphological evidence, Mathias and Constance (1962) concluded that Domeykoa is closely related to Asteriscium, Eremocharis and Pozoa, while Tseng (1967) suggested a close relationship among Domeykoa, Eremocharis, Asteriscium, Gymnophyton, Mulinum and Diposis. The species of Hermas (Figs. 3133) are also similar to these genera in their fruit anatomy, having triangular mericarps and prominent lateral ribs or wings. The large genus Azorella (c. 70 spp., see Pimenov and Leonov 1993; Martinez 1993b) exhibits significant variation among its species (Martinez 1989, 1993a, 1993b). Tseng (1967) showed that Az.cryptantha Clos and Az. spinosa (Ruiz & Pav.) Pers. have lateral ribs that are more prominent than the marginal ribs, precisely as in the sample of Az. incisa studied here (Fig. 6). Furthermore, the mericarps of Az. cryptantha and Az. spinosa are more or less triangular and are thus very similar in shape to those of the other genera listed above. Tseng (1967) suggested that Schizeilema and Spananthe are closely allied to Azorella and Laretia, echoing an earlier view expressed by Reiche (1901). Henwood and Hart (2001) stated that the pollen grains of Azorella, Laretia and Schizeilema share the presence of an ectoapertural bridge. Our results show that the mericarp shape of Schizeilema (Figs. 50, 51) and Spananthe (Fig. 52) is similar to that of some species of Azorella (Figs. 4, 6, 7) and Laretia (Fig. 40), but not to Azorellatrifurcata, Az. compacta, Az. corymbosa (Figs. 3, 5, 8) and Laretiaacaulis (Fig. 39). The diversity of wing types in Azorella and Laretia suggests that the two genera may not be monophyletic, a conclusion that was also reached by Chandler and Plunkett (2004) and Andersson et al. (2006) based on molecular sequence data, suggesting that a detailed phylogenetic study of Azorella and related genera may yield results of taxonomic significance. Henwood and Hart (2001) primarily used fruit morphological and anatomical characters in their preliminary phylogenetic study of Australian genera of “hydrocotyloids”. Their results indicated that Oschatzia may be close to the Bowlesia clade, but our result showed that this genus may be close to Gymnophyton in carpophore structure.

Tseng (1967) showed that the arrangement of the innermost endocarp fibers varies in the different genera of Drude’s Hydrocotyloideae. In all the above taxa, the cells of the innermost layer around the seed are invariably longitudinally orientated (e.g., Figs. 58, 62), which differs obviously from the other taxa below. Therefore, these genera are considered to be closely related and the fiber direction in the fruit may be of taxonomic significance.

Group 2. Taxa with marginal wings and an endocarp with the innermost layer of fibres mainly transversely arranged (Choritaenia only)—The combination of carpological features found in Choritaenia is unique for the genus (Liu et al. 2007a), including the presence of surface trichomes, marginal wings, a very broad commissure, oil vesicles, an endocarp not extending into the wings and a total absence of crystals. The mesocarp is partially woody, a state also found in some genera of subfamily Apioideae (e.g., Coriandrum L., Krubera Hoffm. and Semenovia Regel & Herder). In Apioideae splitting of vascular bundles in the marginal ribs of Ducrosia Boiss. and Lomatium Raf. (personal observation) is considered similar to that in Choritaenia. The fruits of Choritaenia clearly differ from those of all genera currently included in Azorelloideae. Future phylogenetic studies are needed to clarify its position.

Group 3. Taxa with the woody endocarp thickened in the ribs and an endocarp with the innermost layer of fibers arranged in different directions (Klotzschia only)—Shoup and Tseng (1977) studied the pollen of Klotzschia and reported similarities with several genera of Araliaceae (e.g., Eleutherococcus Maxim., Reynoldsia A.Gray, Schefflera J.R.Forst. & G.Forst., and Tetraplasandra A.Gray). The structure of the woody endocarp is the same in Klotzschia as in some members of Araliaceae, e.g., Acanthopanax Miq., Eleutherococcus and Macropanax Miq. The results of molecular systematic studies (reviewed in Plunkett 2001), however, have shown that Klotzschia forms a separate lineage between the apiaceous clades now recognized as subfamilies Mackinlayoideae and Azorelloideae, which prompted Plunkett et al. (2004) to exclude Klotzschia from Azorelloideae. This decision is supported by the fruit anatomy of Klotzschia, which is distinctly different from that of all other taxa examined here.

Other characters. Rib secretory ducts, vittae and vesicles

The presence and nature of secretory canals have played a prominent role in the phylogenetic speculations of Baumann (1946), Tikhomirov (1961), and Eyde and Tseng (1971). In the taxa studied here, rib secretory ducts are present in almost all genera (e.g., see Fig. 3), but they also characterize Araliaceae, and the apiaceous subfamilies Mackinlayoideae and Saniculoideae sensu lato, as well as some Apioideae. Eyde and Tseng (1971) suggested that “the gynoecia of ancestral Araliaceae were well supplied with scattered secretory canals, that the canals have been lost in a few of the derived taxa, and that they have been localized in others, including the Umbelliferae [=Apiaceae].” Secretory vesicles were found only in Choritaenia, Bilacunaria Pimenov & V.N.Tikhom. and Smyrniopsis Boiss. (subfamily Apioideae) (Tamamschan 1946; Liu 2004; Liu et al.2007a). Elsewhere in Apiales, similar vesicles occur only in the genera of Myodocarpaceae (Myodocarpus and Delarbrea; personal observation, and see Lowry 1986a, b). Reticulate and irregular vittae are present in Dickinsia (Liu et al. 2002), Hermas and Klotzschia but this character also occurs in several species of the Apioideae and therefore seems to have arisen independently in different lineages.

Ventral bundles and carpophore

Ventral bundles are the vascular tissue associated with the commissure. Two ventral bundles, as exemplified by those of Mackinlaya confusa (Fig. 1), are widely present in members of subfamily Mackinlayoideae (e.g., Centella L. and Micropleura Lag.). An entire carpophore occurs commonly in subfamily Azorelloideae (e.g., Fig. 60). Most apioid taxa, however, have a carpophore splitting into two halves, as in Bupleurum and Heteromorpha Cham. & Schltdl., and this feature also characterizes the azorelloids (e.g., Diposis saniculifolia, Gymnophyton robustum, Figs. 20, 26). The type of carpophore characteristic of Gymnophyton isatidicarpum (Fig. 28), Oschatzia cuneifolia and O. saxifraga (Figs. 46, 47), is also found in Astydamiacanariensis (Spreng.) DC. (subfamily Apioideae). A very short carpophore as in Choritaenia capensis has not been found in any other taxa of the family (Liu et al. 2007a). A carpophore is usually absent in Araliaceae (except in Astrotricha DC. and Trachymene Rudge) and in Apiaceae subfamilies Mackinlayoideae and Saniculoideae (except in Alepidia Delar.). A detailed study of carpophore structure in the Azorelloideae is currently underway.

Crystal type and distribution

Two main types of crystals are found in the Apiaceae and Araliaceae, namely rhomboidal crystals and druse crystals. Rhomboidal crystals (called cyclic crystals by Kljuykov et al. 2004) (Fig. 58) are present as individual structures in a single cell layer of the mesocarp and were observed in almost all of the genera studied. This type of crystal also characterizes Apiaceae subfamily Mackinlayoideae and some Araliaceae (e.g., Hydrocotyle and Trachymene) but they are totally absent from all taxa of the subfamilies Saniculoideae and Apioideae. Druse crystals are compound structures, more or less spherical in shape, that are typically dispersed throughout the mesocarp. The crystals are often dissolved during sample preparation, leaving characteristic “ghost cells”. Druse crystals are absent from all the taxa studied in this paper. They are typically found in the subfamilies Saniculoideae and Apioideae. If present, they are usually amphi-seminal (scattered around the seed) (most Saniculoideae, e.g., Sanicula L. and Eryngium L.) or present only on the commissural side of the seed (some Apioideae, e.g., Myrrhis odorata (L.) Scop. and Astrodaucus littoralis Drude). Druse crystals are also found in Araliaceae (e.g., Eleutherococcus brachypus (Harms) Nakai and Brassaiopsis chengkangensis H. H. Hu).

Conclusions

A combination of carpological features, including the woody endocarp, the orientation of the innermost fiber layer of the endocarp and the usually triangular mericarps bearing prominent lateral ribs or wings, largely confirms the concept of Apiaceae subfamily Azorelloideae, as suggested previously by molecular analyses (Plunkett et al. 1996, 1997, 2004; Downie and Katz-Downie 1999; Downie et al. 1998, 2001; Plunkett 2001; Chandler and Plunkett 2004) and fruit anatomical studies (Liu 2004). On the basis of the DNA results, a new subfamily was formally recognized by Plunkett et al. (2004) as Azorelloideae Plunkett & Lowry. The fruit anatomical data presented here reinforce and expand the conclusions of these earlier studies by including a considerably broader sampling. Based on anatomical evidence, we suggest that all of the taxa placed in tribe Mulineae by Pimenov and Leonov (1993) should be included in Azorelloideae, with just two exceptions, Choritaenia and Klotzschia, which together comprise only four species. Regarding Azorella and Laretia, the broad morphological variability observed in the fruits of these genera appears to support the finding that both genera may not be monophyletic, as suggested by Chandler and Plunkett (2004) (see also Andersson et al. 2006). Recent taxonomic revisions are available for Azorella (Martinez 1993b) and Mulinum (Zech 1992), but Chandler and Plunkett (2004) found that Azorella was paraphyletic with respect to both Mulinum and Huanaca. It is clear, therefore, that these genera must be studied in the broader context of the entire subfamily, and future molecular studies should explore the phylogenetic positions of these and other azorelloid taxa. Perhaps most importantly, in the wake of the collapse of the traditional systems of classification of Apiaceae, which have proven inadequate when compared to findings based on molecular data, the present study and other recent analyses based on fruit anatomy (Liu 2004; Liu et al. 2003, 2006, and unpublished data) hold out the promise of identifying reliable features that can be used to erect a more stable classification of the entire family that reflects underlying evolutionary relationships, something that has eluded generations of botanists.

Acknowledgments

We gratefully acknowledge the curators and staff of BOL, CBG, CHR, F, GH, K, MO, NAS, NE, NSW, NY, PRE, RAB, S, SP, U, VCU for supplying fruit samples or for permission to collect fruits for our study. Financial support was provided to ML and BEVW from the University of Johannesburg and the National Research Foundation (South Africa), from the US National Science Foundation to GMP (DEB 0613728) and to PPL (DEB 0614152) and from the China National Science Foundation to ML (30870148).

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