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Home , Antirrhineae, Asarina, Chaenorhinum, Chelone (plant), Cymbalaria, Galvezia

Plant Syst. Evol. 220:223-239 (2000) Systematics and Evolution © Springer-Verlag 2000 Printed in Austria

Phylogeny of the tribe () based on morphological and ndhF sequence data

Medhanie Ghebrehiwet 1, Birgitta Bremer 2, and Mats Thulin 2

1Department of Biology, University of Asmara, Asmara, Eritrea 2Department of Systematic Botany, Uppsala University, Uppsala, Sweden

Received November 5, 1998 Accepted May 18, 1999

Abstract. Phylogenetic relationships within the Key words: Scrophulariaceae, Antirrhineae, mor- tribe Antirrhineae (Scrophulariaceae) are analysed phology, ndhF, phylogeny, pollination. and discussed on the basis of parsimony analyses of morphological and ndhF gene sequence data. The tribe Antirrhineae as revised by Sutton The results indicate that the tribe Antirrhineae (1988) consists of 27 genera representing 328 consists of four major groups of genera, the species. Familiar members include various , the Gambelia clade, the Maur- garden ornamentals, such as snapdragons andya clade, and the clade. The (Antirrhinum), toadflax (), and kenil- Anarrhinum clade, consisting of the Old World worth (). The group is a clearly bee-pollinated genera Anarrhinum and , is sister to the rest of the tribe. The Gambelia clade circumscribed tribe in the Scrophular- consists of the New World genera Gambelia and iaceae and is characterised, above all, by its , which are very closely related and poricidal dehiscence, unique in the pollinated by . The family, and by the presence of apparently clade consists of one subclade including Maur- unique iridoid glycosides, such as antirrhino- andya and a number of related bee- or humming- side (Kooiman 1970). Preliminary studies of bird-pollinated New World genera and another morphological and rps2 sequence data by subclade with the Old World bee-pollinated genera Depamphilis et al. (1994) indicate strong and Cymbalaria. The Antirrhinum clade support for the monophyly of the Antirrhineae. consists mainly of bee-pollinated Old World The relatively stable taxonomic history of the genera, such as Antirrhinum, Linaria, Chaenorhi- Antirrhineae is a reflection of the marked num, and their segregates, but also includes the New World genera and Howelliella, of internal continuity and external discontinuity which the latter is known to be partly pollinated by of its morphology. Its circumscription has hummingbirds. It is concluded that - remained more or less the same whether it was pollination has evolved independently within treated as a single (Linnaeus 1753), as a Antirrhineae at least three times from bee-polli- tribe (Chavannes 1833), or as a subfamily nated ancestors. (Betsche 1984). 224 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae)

The supra-genetic relationships within the Maurandyinae have several derived characters Antirrhineae have been discussed by, e.g., that will place its members in a relatively Rouy (1909), Rothmaler (1943), and Speta advanced position in the tribe, though his view (1982). Mainly on the basis of the palate was not supported by others (Sutton 1988, structure (the convexity of the abaxial Thompson 1988). In his revision of the tribe lip) Rouy (1909) classified the Antirrhineae Antirrhineae Sutton (1988) made some com- into three subtribes, Linariinae (as "Linar- ments and remarks of disagreement with ieae"), Anarrhininae (as "Anarrhineae"), and Elisens' work, suggesting the need for a Rhodochitoninae (as "Rhodochitoneae"). detailed phylogenetic study of the whole tribe. with a prominent palate were put in Recent studies based on molecular data the Linariinae (Linaria, Antirrhinum and have begun to unravel relationships within the ), and those without a clearly family Scrophulariaceae that will also give defined palate into Anarrhininae (most genera some clues about the position of the Antir- including Anarrhinum and Maurandya). Rho- rhineae in the family. These include the dochiton was treated as a separate subtribe on studies by Depamphilis et al. (1994) and the basis of its unique membranaceous calyx. Olmstead and Reeves (1995), who have Rothmaler (1943) divided the tribe into demonstrated the polyphyly of the family. five groups: "Maurandya-Gruppe"(five gen- The results obtained by Olmstead and Reeves era), "Gambelia-Gruppe" (three genera), (1995) by using rbcL and ndhF gene "Linaria-Gruppe " (11 genera), "Mohavea- sequences indicate that the Scrophulariaceae Gruppe" (Mohavea only), and "Anarrhinum- are composed of two distinct desig- Gruppe" (Anarrhinum only). These groups nated by the authors as "Scroph I" and have been treated later on by Rothmaler "Scroph II". The Antirrhineae represented himself (Rothmaler 1954) and subsequent by Antirrhinum is shown to fall within authors as the subtribes Maurandyinae, Gam- "Scroph II". This clade further includes beliinae, Linariinae, Mohaveinae and Anar- and from Scrophulariaceae rhininae (as "Simbuletinae" by Rothmaler, as well as representatives of , 1954). Rothmaler (1943) further attempted to Callitrichaceae, and Hippuridaceae. Recently, show phylogenetic relationships between the it has been shown that also Globularia belongs genera within the tribe. He assumed that to this clade (Oxelman et al. 1999). palmate venation was a relatively primitive The floral diversity within Antirrhineae is character state, while development of palate considerable and includes for example corollas and spur were amongst the derived states. He closed by a palate, narrow tubular corollas, wide placed the "Maurandya-Gruppe" in a basal flaring corollas, and corollas with a spur. position on one of the main branches on the of the Old World species of the tribe tree with nearest the root. The (including Anarrhinum, Antirrhinum, Asarina, "Anarrhinum-Gruppe" was placed as a sister , Cymbalaria, Kickxia, Linaria, to all other genera of Antirrhineae. and ) are normally zygomorphic, but Rothmaler's classification and evolution- many of the New World Antirrhineae (particu- ary interpretation has been changed and larly , Maurandya, and Rhodo- criticised by several subsequent workers. chiton) have peloric flowers with corollas that Speta (1982) confined the Linariinae to approach the actinomorphic state (Sutton 1987). Linaria only, whereas the remaining genera In the Antirrhineae most of the species are bee- were placed in the new subtribe Antirrhininae. pollinated, but pollination by hummingbirds Rothmaler's evolutionary interpretation on the is known from several New World members status of the Maurandyinae was rejected by of the tribe (Pennell 1935, Elisens 1986, Sutton Elisens (1985a), who suggested that the 1988). M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 225

The aim of the present work is to present a 1994) analyses were performed. Bootstrap was hypothesis for the phylogenetic relationships calculated from 1000 replicates, each with a single, within the tribe based on morphological data random additional sequence of the taxa and for all the genera recognised by Sutton (1988) nearest-neighbour interchanges (NNI) branch swap- and complemented by molecular data for a ping saving a single tree. Bremer support is defined as the number of extra steps necessary to lose a selected sample of genera, and to discuss group in the consensus. Character evolution was trends in floral evolution and pollination traced using the computer program MacClade systems within the tribe, as well as previous (Maddison and Maddison 1992). subtribal classifications, in the light of this Morphological characters. Several of the phylogenetic hypothesis. characters and codings require some additional comments. Polymorphisms are coded by all observed states but, the widely common state is Materials and methods used when there is an absolute majority for one of . In all analyses Digitalis and the states (see Weins 1995). Unknown character were used as outgroups. Chelone was selected on states are coded with a question mark. In order to the basis of ongoing research suggesting it to be minimise unknown states in characters inapplic- close to the Antirrhineae (Eberhardt Fischer, pers. able for taxa lacking particular organs, absence of comm.). A common origin of Cheloneae and organs was included as a state in a multistate Antirrhineae is also suggested by Raman (1990) character (Maddison 1993, Swenson and Bremer on the basis of similarity in the trichomes present 1997). Coding of quantitative characters was done on the corolla. Digitalis was selected as a member following Thiele (1993), who in contrast to Stevens of the "Scroph II" clade of Olmstead and Reeves (1991) supports the applicability of overlapping (1995) along with Antirrhinum. morphometric data in phylogenetic analyses. Morphological analysis. A data matrix was Habit. In their habit the Antirrhineae are prepared comprising 30 taxa and 30 characters. All typically herbs, but some are subshrubs or small genera recognised by Sutton (1988) were included shrubs. The stems may be erect, procumbent or in the analyses. The genera were used as terminals climbing. Approximately half of the genera of the in all cases except Kickxia, where the two sections Antirrhineae includes only perennial species. K. sect. Kickxia and K. sect. Valvatae were used to . The shape of the leaves is quite be able to test the monophyly of the genus. These variable in the Antirrhineae and often diagnostic two sections show marked differences and a at generic level (Sutton 1988). The range of shape subdivision into distinct genera has been proposed is coded here in three states as isodiametric leaves, by Betsche (1984). The information on the elongate leaves, and linear to filiform leaves. characters is based on literature (Sutton 1988, Isodiametric leaves include such with deltate, Elisens 1985a, Thompson 1988) supplemented by hastate, sagittate, orbiculate, or reniform shape, studies of herbarium material of all genera and while elongate leaves may be elliptic, ovate, cultivated material of many taxa. The various obovate, oblanceolate, or oblong. shape in morphological characters used are listed in Table 1 the Antirrhineae may vary considerably in different and the data matrix is presented in Table 2. parts of the plant. Basal leaves are in some genera The data matrix was analysed using the often markedly different in size and/or shape from computer program PAUP version 3.1.1 (Swofford the leaves higher up on the stem, i.e. the leaves are 1993). The parsimony analysis was carried out heteromorphic. The homomorphic condition is using heuristic methods and with all character coded as one state and the heteromorphic condition changes weighted equally. The options used were as two states, different in size and different in TBR branch swapping with MULPARS on, shape. steepest descent off and random addition Rothmaler (1943) sometimes used the venation sequences with 100 entry replicate searches. All pattern of the leaves in his delimitation of genera in multistate characters were treated as unordered. To the tribe. Palmate venation is prevalent in the assess the relative support for clades bootstrap genera with more or less isodiametric leaves. Most (Felsenstein 1985) and Bremer support (Bremer genera in the Antirrhineae have entire or lobed 226 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae)

Table 1. Characters and character states used in the morphological analysis of Antirrhineae

. Habit herbaceous (0), suffrutescent (1), shrubby (2) 2. Duration perennial (0), annual/biennal (1) 3. Stem erect (0), procumbent or climbing (1) 4. Upper and lower leaves homomorphic (0), different in size (1), different in shape (2)

. Leaf shape isodiametric (0), elongate (1), linear to filiform (2) 6. Leaf margin dentate (0), lobed or entire (1) 7. not cirrhose (0), cirrhose (1) absent (2) 8. Venation pinnate (0), palmate (1) 9. Pedicel not cirrhose (0), cirrhose (1) 10. Calyx lobes equal (0), adaxial lobe longest (1), adaxial lobe shortest (2) 11. Calyx shorter than corolla tube (0), longer than corolla tube (1) 12. Corolla up to 20mm long (0), more than 20 mm long (1) 13. Corolla tube up to 5 mm wide (0), more than 5 mm wide (1) 14. Corolla tube length < 2x width (0), >_2x width (1) 15. Corolla tube not spurred (0), spurred (1) 16. Corolla lips equal (0), adaxial lip shorter (1), adaxial lip longer (2) 17. Abaxial lip without palate (0), with palate (1) 18. Anthers free (0), marginally coherent (1), forming ring (2) 19. Staminode with rudimentary anther (0), without rudimentary anther (1) 20. Shape of capsule ovoid to subglobose (0), globose (1), oblong (2) 21. Loculus size of capsule equal (0), unequal (1) 22. Capsule wall coriaceous (0), papery (1) 23. Dehiscence by single split (0), valvate (1), irregular rupture (2) 24. Seed symmetry radial (0), bilateral (1), dorsiventral (2) 25. Position of hilum basal or subbasal (0), median (1) 26. Seed size> 2mm (0), 1 -2mm (1), < lmm (2) 27. Wing of seed of Epixiphium type absent (0), present (1) 28. Tubercles absent (0), present (1) 29. Periclinal wall of testa cell flat (0), convex (1), concave (2) 30. Epicuticular wax absent (0), present (1)

leaves, but in some the margin is dentate or (1943). The character is here coded as calyx lobes crenate. shorter or longer than corolla-tube. The presence or absence of a petiole is an Corolla. The corolla of most Antirrhineae is important character in the delimitation of Antir- gamopetalous, 5-lobed, tubular and bilabiate, and rhineae genera. The petiole can be cirrhose or not. the abaxial lip has a basal convexity, the palate, All species of Linaria have sessile leaves. which occludes the mouth of the tube (Sutton Pedicel. The flowers in Antirrhineae are 1988). The floral tube is variable in length, width, pedicellate, and the pedicel can be cirrhose or and shape. The abaxial base of the tube is known to not. Only in few species the pedicel is virtually exhibit different shapes variably known as entire, absent. saccate, gibbous, or spurred (Sutton 1988). There is Calyx. The calyx is normally deeply divided no clear-cut separation between these shapes as into five lobes. A few taxa, Rhodochiton and one one type continues to the other. Only two states are species of Anarrhinum, have a shallowly lobed used here, spurred and not spurred. In Antirrhinum calyx. The relation between the length of the calyx the corolla-tube is generally gibbous, but one to that of the corolla-tube has been used as a species, Antirrhinum braun-blanquetii, is spurred. diagnostic character at genetic level by Rothmaler In Kickxia, on the other hand, all species except M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 227

Table 2, Data matrix for the terminal taxa (with names) and the characters (numbered as in Table 1). Polymorphic states are represented by letters as follows: a = 0/1, b = 0/2, c = 0/1/2, d = 1/2. Unknown and inapplicable states are coded "?"

1 1111111112 2222222223 ~xa 1234567890 1234567890 1234567890 Digitalis 0001100000 01100000?0 00?1 01 00?0 Chelone 0000100000 011000a000 00?0001000 Acanthorrhinum 2000212000 0000021111 10110101 a l Albraunia 0100110002 1000101112 00000101al Anarrhinum 0002100000 0000111210 0a11120110 Antirrhinum c000bl0000 0a11021110 1010020011 Asarina 1010000100 0111001110 0110010000 Chaenorhinum aaO0 l10001 00001011 lc 1 a 10020111 Cymbalaria 0al0010101 0000101111 01100c01al Epixiphium 0010011100 0111000100 1012001101 Galvezia 2000110000 0a010 010 00 0120020100 Gambelia 2000110000 01 al01101"0 10010d0a 10 1000000101 0111001001 1120000100 Holzneria 0100110002 1000101110 101002001 a Howelliella 0100110001 0a01111100 1120020010 Kickxiasect. K. ca12110100 0000111211 0a01120a10 Kickxiasect. V. cal2cl0100 00a0111210 0al 1120110 Linaria 0aaa212 00c 00a01011 lc al lb0a?ac0 Lophospermum 1011001110 0111010001 0122001120 1000000100 0111010000 0120010120 Maurandella 1011011100 0111011 lOb 1121010101 Maurandya 1011011111 0111010102 01200101?0 Misopates 0100110001 0001011110 1011021 lal Mohavea 0100110001 0101001100 1111011000 0102110010 OOaO011111 a0200201 O0 0100112000 0001111112 01100201?0 0100110000 00010111 O1 01110011 O1 Rhodochiton 1010001100 0111000002 0122001100 0100110001 0001011 llb 11100101?? Schweinfurthia la00al0 001 0000001111 1120010111

K. saccata are spurred. Antirrhinum is coded here shapes are globose, ovoid, and oblong. The capsule as not spurred, while Kickxia is coded as spurred. is bilocular and in most genera the loculi are equal, . Normally the Antirrhineae have four but a few have conspicuously unequal loculi with fertile stamens with the fifth (adaxial) the abaxial loculus usually the largest. The loculus reduced to an inconspicuous staminode with or wall can either be thin and papery or thick and without a rudimentary anther. In the majority of the coriaceous. Although all of the Antirrhineae genera the stamens are conspicuously didynamous. basically have a porose pattern of dehiscence, the Commonly the adjacent stamens are coherent by mode of dehiscence can be by a single split, the margins of the anthers, but in Kickxia and valvate, or by irregular rupture. Anarrhinum all four anthers are connate, forming a Seeds. The seeds of Antirrhineae show a great ring-like structure. variation in size, shape, and external ornamenta- Capsule. The capsules of Antirrhineae vary tion. This variation has been used by several markedly in size and shape. The most common authors (Munz 1926, Rothmaler 1943, Elisens 228 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae)

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1985b, Sutton 1988) for generic and infrageneric data matrix in the phylogenetic analyses comprise delimitation within the tribe. The size of the seeds 2133 aligned nucleotide positions. in the tribe varies from c. 4 mm in genera such as The parsimony analysis was carried out using Holmgrenanthe, Lophospermum, and Rhodochiton heuristic methods and with all character changes to c. 0.3 mm in some sections of Linaria and in weighted equally. The options used were the same Nuttallanthus. An average size, as exemplified by as in the morphological analysis. species of Maurandya, is between 1 and 2 mm. Combined analysis. The molecular and mor- In the majority of the genera the position of the phological data for the same terminal taxa as in the hilum is basal or sub basal, but also a median molecular analysis were merged into one data position is sometimes found. Sutton (1988) has matrix with a total of 2163 characters. The indicated that the wings of the seeds of Linaria, parsimony analysis was carried out using heuristic when compared in relation to the position of hilum, methods and the options used were the same as in are not homologous to those of Epixiphium, the previous analyses. Lophospermum, Rhodochiton, Mohavea, and Pseud- orontium. The symmetry of the seeds" in Antir- rhineae is variable, and three types have been Results distinguished by Sutton (1988), radial, bilateral, Morphological analysis. The cladistic analy- and dorsiventral. sis of the morphological data with 30 char- The seeds of Antirrhineae possess an intact outer periclinal wall to the testa-cells at maturity. acters and 30 taxa generated 134 equally most The curvature of the outer periclinal wall is parsimonious trees all with a length of 140 responsible for the often microscopically visible steps. The consistency index is 0.300 and the roughness of the seed surface. Individual testa-cells retention index 0.616. Within Antirrhineae may have the periclinal wall flat, convex, or two major clades appear in the strict consensus concave (Sutton 1988). Epicuticular secretions on tree (Fig. 1). The first clade is composed of seeds usually occur as irregular to very regular New World genera that belong to the subtribe striations with a high micromorphological diversity Maurandyinae (Rothmaler 1954, Elisens (Barthlott 1981). In the Antirrhineae several genera 1985a) with the Old World genus Asarina have seeds with an overlaid fine texturing of nested within it. The second clade is less granules or filaments of epicuticular waxes (Sutton resolved, with Galvezia coming out as the sole 1988). In the analysis this character is coded as sister to the rest. The subclade containing present or absent. Molecular analysis. Sequences from 19 term- Anarrhinum and the two sections of Kickxia is inal taxa, including outgroups and the two sections strongly supported. of Kickxia, were used for the cladistic analysis of Molecular analysis. The molecular search molecular data. The species used in the analysis resulted in two equally most parsimonious are listed in Table 3, with details of their name, trees, 977 steps long with a consistency and voucher specimens and EMBL accession numbers. retention index of 0.735 and 0.634 respec- Most of the plant material used for extracting DNA tively. The results indicate that Antirrhineae was grown from seeds, but in a few cases can be subdivided into four well supported herbarium material was used. DNA was extracted, groups of genera (Fig. 2). The first group, amplified, and sequenced following the protocols corresponding to the Anarrhinum clade includ- in Kim and Jansen (1995) or Backlund et al. ing Anarrhinum and the two sections of (in press). Alignments were made manually to the Kickxia, is sister to the rest of the Antirrhi- reading frames of the corresponding protein sequence, gaps were treated as missing data. In neae. This clade is highly supported with a the data analysis 15 sequences of species repre- bootstrap value of 100 and a Bremer support senting genera in the Antirrhineae and the one of of 27. The two sections of Kickxia are sister Chelone are new, while the sequences for Antir- groups with Anarrhinum as sister to the genus rhinum and Digitalis were already published. The Kickxia. 230 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae)

Digitalis -] Chelone outgroup Holmgrenanthe Asarina 1 Mabrya Maurandella 1 ' Maurandya 1 Epixiphium Lophospermum 84 1 Rhodochiton 4 Galvezia Chaenorhinum Cymbalaria Gambelia Howelliella

1 Linaria Misopates Mohavea Neogaerrhinum Nuttallanthus

1 Pseudorontium Sairocarpus Schweinfurthia Acanthorrhinum Fig. 1. Strict consensus tree of the 11 lJ Antirrhinum equally most parsimonious trees of the 55[ Albraunia Antirrhineae based on morphological Holzneria characters. Numbers above nodes corre- I spond to bootstrap values above 50%. 76 Anarrhinum Numbers below nodes are Bremer support Kiekxia sect. K. values, i.e. numbers of extra steps required Kickxia sect. V. to collapse that particular node

The second group is the Maurandya clade contains the two shrubby genera Gambelia and that consists of one subclade including the Galvezia from Central and South America. New World Maurandyinae sensu Elisens This clade has a bootstrap support value of (1985a) and sister to it another subclade with 100 and a Bremer support of 23. The rest of the Old World Asarina and Cymbalaria. The the genera, including Chaenorhinum, Linaria, clade is well supported with a bootstrap value Antirrhinum, Misopates, Schweinfurthia, of 84 and a Bremer support of 6, and all the Howelliella, and Mohavea, make up the subclades are also well supported (Fig. 2). Antirrhinum clade, which has a bootstrap The remaining genera are in a large clade support of 80 and a Bremer' support of 5 with a bootstrap value of 55 only, but it is (Fig. 2). possible to divide it into two well supported Combined analysis. The combined anal- subclades. One of these, the Gambelia clade, ysis of the morphological and molecular data M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 231

DigitaliSchelone 10utgroup

Anarrhinum 10o - Anarrhinum 27 83 1 Kickxiasect. K. I clade 5 1 Kickxiasect. V. Asarina 94 Cymbalaria 2I 84 Rhodochiton Maurandya 92 clade lO 93 ~. Maurandella 8_.9._0 11 Maurandya 4 Galvezia lOO i] Gambelia Gambelia clade 55 1 Chaenorhinum 805~ [ Linaria Fig. 2. Strict consensus tree of the Antirrhinum two equally most parsimonious trees Antirrhinum of the Antirrhineae based on molec- / 100] Misopates Clade ular (ndhF) characters. Numbers 22199t--Schweinf urthia above nodes correspond to bootstrap values above 50%. Numbers below 79 L Howelliella nodes are Bremer support values, i.e. numbers of extra steps required to 1 Mohavea collapse that particular node

resulted in two most parsimonious trees both morphological characters have been optimised 1085 steps long, with consistency and reten- on one of the combined trees in Fig. 4. tion indices of 0.698 and 0.613 respectively. Presumed (information from Grant The strict consensus tree is shown in Fig. 3. and Grant 1966, Elisens 1986, Sutton 1988, Except for the collaps of the branch uniting the Thompson 1988, Kampany 1995), which were Gambelia clade and the Antirrhinum clade the not included as characters in the analyses, are topology of the combined tree agrees with that also indicated for each genus on the tree. of the molecular tree. All four major clades are well supported with a bootstrap value of 100 Discussion for the Anarrhinum and the Gambelia clades, 90 for the Maurandya clade, and 74 for the The results from both the molecular and the Antirrhinum clade. The corresponding Bremer combined analyses suggest that-the Antirrhi- support values are 29, 21, 8, and 3. Most of the neae can be subdivided into four main groups subclades are also well supported. The of related genera corresponding to the Anar- 232 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae)

DigitaliSchelone ~l Outgr°up

Anarrhinum 1 100 29 ~ Kickxia sect. K Anarrhinum 88 clade 5 ~ Kickxia sect. V.

100 ~ Galvezia --I Gambelia 99 21 ~ Gambelia _1 clade 23

100 Asarina 12 Cymbalaria 90 Maurandya 58 8 Rhodochiton clade 2 98 12198 -- Maurandella

13 ~ Maurandya

Chaenorhinum --

74 Linaria Fig. 3. Strict consensus tree of the two 3 67 equally most parsimonious trees of the 2 Antirrhinum Antirrhinum Antirrhineae based on a combined data 100 set of morphological and molecular 23 Misopates clade (ndhF) characters. Numbers above 63 2 ~ Schweinfurthia nodes correspond to bootstrap values 92 above 50%. Numbers below nodes are 5 L Howelliella Bremer support values, i.e. numbers of 84[- extra steps required to collapse that 3 Mohavea particular node

rhinum clade, the Gambelia clade, the Maur- the four groups mentioned above, only the andya clade, and the Antirrhinum clade (Figs.- Anarrhinum clade appeared as a distinct group 2, 3). The clades are all well supported with with a bootstrap value of 76 and a Bremer bootstrap values ranging between 74 and 100 support of 1. Although well resolved, the and Bremer support values ranging between 3 Maurandya clade has a low support and, and 29. unlike in the molecular analysis, Cymbalaria The analysis of the morphological data is not included. Nevertheless, in the combined produced trees with low CI and a relatively tree the support for several branches, including poorly resolved consensus tree (Fig. 1). Most that of the Maurandya clade, was increased of the clades in the morphological tree have (Fig. 3) compared to the molecular analysis low support values. It is therefore scarcely (Fig. 2). This indicates that the morphological possible to deduce phylogenetic relationships data to some extent support the results of the from the morphological results alone. Among molecular data. M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 233

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=t= 12:0 [ 1:21:0[ ~121 : 6:1 1:°1 ,'~ f2o:~,o.i:~ 23 k_~ 9:'1 k_. • ~ 28:0I I T 27:1 22 ~I22:01 26:1~ ~" 12:1 6:° I :I=16:1 14:] I0 -27:I-~L---~ 19:0 -23:2 ll:24:1 [ = 20:2 : 22:0 I 19:0 :16:1 I 7:1 30:01 ~14:01 ~10:1 =~2:1

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Fig. 4. Morphological characters optimised on one of the most parsimonious trees of the combined analysis• Black bars indicate synapomorphies with ci = 1, open bars indicate homoplastic characters with subsequent changes, crosses indicate reversals, and double bars indicate parallelisms. Type of pollinators are indicated in bold after each taxon. codes: B bee; H hummingbird; M moth

An interesting result in the molecular and pointed to by Pennell (1947), who even treated combined analyses is the appearance of the the two taxa as congeneric. Opposing Pen- Old World Cymbalaria and Asarina as sister nell's view Elisens (1985a, 1985c) rejected a taxa to the New World Maurandya and its close relationship between Asarina and Maur- relatives. In his Rothmaler andya and its relatives, and selected the New (1943) placed Asarina and Cymbalaria close World Neogaerrhinum as an outgroup taxon to to each other and as an offshoot of the detect phylogeny in the Maurandyinae. Maurandya group. The close relationship In the present molecular and combined between Asarina and Maurandya was also analyses the support for the Maurandya clade 234 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) is strong. Isodiametric leaves is a synapomor- Antirrhineae in the molecular and combined phy for this clade. This is a character analyses, are all bee-pollinated with basally otherwise found only in certain members of spurred corolla-tube and with the mouth Kickxia sect. Valvatae. Also, Sutton (1988) partially closed by a palate. When compared stated that a high proportion of radially to the outgroup, these floral characteristics of expanded epidermal cells give rise to the the Anarrhinum clade can be considered as swollen, multicellular tubercles and ridges derived. Both groups are bee-pollinated but present in both Maurandyinae, Cymbalaria show differences in the strategy of attracting and Asarina, but as data for many of the particular pollinators. The palate functions as genera were not available, this character was a barrier to ineffective visitors, and only not included in the analysis. strong, heavy, long-tongued bees are capable Trends in floral evolution. Most members of reaching the . In Chelone and of the family Scrophulariaceae have flowers Digitalis, on the other hand, the corolla-tube with a five-lobed calyx, a campanulate corolla is very wide and allows pollinators to freely with a slightly gibbous corolla-tube bearing move inside. It should be noted also that there five lobes, four stamens attached to the inside are close relatives of Chelone and Digitalis of the corolla-tube, and a bicarpellate pistil that are pollinated by hummingbirds, butter- (Kampany 1995, Kampany and Dengler 1997). flies, or flies (Kampany 1995). The occurrence of this type of in most The floral diversity is considerable in the tribes of the Scrophulariaceae and related Maurandya clade. The flower size ranges from families indicate that it is the basic (ancestral) 6 to 15ram in Cymbalaria and from 45 to form of flower in the group (Kampany 1995). 55 mm in Rhodochiton. The corolla-tube is The two outgroup species and variable in length, width, as well as in shape. It Digitalis grandiflora share all the ancestral is abaxially spurred in Cymbalaria, gibbous in floral characteristics. Some close relatives of Maurandya and entire in Rhodochiton. This Chelone have the corolla-tube closed by a variation in floral structure is directly related palate (Kampany 1995). to the various pollination systems of the Studies on evolution of floral morphology genera. Members of Cymbalaria, Asarina, in the "Scroph II" clade by Reeves and and Maurandella, have snapdragon-like flow- Olmstead (1998) suggest a common ancestry ers, with a conspicuous palate closing the for Digitalis and Antirrhinum. It was also mouth until depressed by a bee of suitable size suggested that Chelone and Collinsia, both of and weight. Similar but slightly larger are the which are members of the tribe Cheloneae, are flowers of Maurandya and Epixiphium. basal members of the "Scroph II" clade. It is Among the large-flowered members are Rho- likely, therefore, that floral evolution in the dochiton and Lophospermum, which are bird- "Scroph II" clade has taken two main lines. pollinated and have corollas with a gaping One of these is the Digitalis--Calli- throat (Elisens 1986, Sutton 1988). The triche line discussed by Reeves and Olmstead present results agree with Pennell's remark (1998), involving various reductions in floral on the floral evolution of the Maurandyinae parts. The second line is the floral evolution in from bee-pollinated ancestry into enlarged and the Antirrhineae as discussed here. In the reddened corollas fitted to the visits of hum- Antirrhineae, the flower has evolved to form mingbirds (Pennell 1935). corollas closed by a palate, narrow tubular Compared to the other clades, the flowers corollas, wide flaring corollas, and corollas in the Gambelia clade have a uniform with a spur (Pennell 1935, Kampany 1995). structure. In both Galvezia and Gambelia the The members of the Anarrhinum clade, flowers are trumpet-shaped with a long, which appeared sister to the rest of the narrow corolla-tube somewhat swollen at the M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 235 base. They are red in colour and are reported Characters related to pollination systems to be hummingbird-pollinated (Grant and are reported to be labile due to selective Grant 1966). pressure by pollinators resulting in repeated The floral diversity in the Antirrhinum parallelisms and reversals (Armbruster 1992, clade is comparable to that of the Maurandya 1993; Johnson et al. 1998). In Antirrhineae clade. The corollas vary from strongly bilat- this is true particularly for the evolution of eral with the tube closed by an enlarged palate, spur and palate. as in Linaria and Antirrhinum, to corollas In the Antirrhineae nectar is presented to with a gaping throat, as in Howelliella and the pollinator in the abaxial base of the tube Mohavea. The corolla-tube varies from which is either gibbous, saccate or spurred spurred in Linaria to gibbous in Antirrhinum (Elisens and Freeman 1988). The present and saccate in Mohavea. Linaria and Antir- results indicate that a spur has evolved several rhinum provide classical examples of flowers times in the Anarrhinum, Maurandya and specialised for bee-pollination. Mohavea was Antirrhinum clades as a result of parallelisms by Sutton (1988) considered to be the most and repeated reversals. All members of the unusual genus in the tribe as the lips of the Anarrhinum clade are spurred, whereas Cym- corolla are greatly enlarged and only the two balaria is the only spurred member of the abaxial stamens are fertile. The orientation of Maurandya clade and both Gambelia and the flower is also different from that of the Galvezia are without spur. In the Antirrhinum other genera in the Antirrhineae, as the tube is clade both spurred and non-spurred taxa are nearly erect pointing the mouth upwards rather present. The spur is reported to be sometimes than more or less horizontal, and suggesting a very variable within species or genera. In quite different pollination strategy (Sutton , variants with no spur occur 1988). The pollination strategy of Mohavea in addition to occasional mutants with five as reported by Little (1983) involves floral spurs (Sutton 1987). In Kickxia sect. Valvatae food deception mimicry based upon morpho- a single species without spur has been logical similarities of the flowers to that of the described (K. saccata), and Antirrhinum proposed model species braun-blanquetii is the only spurred species (Loasaceae). The only known pollinators for in the otherwise gibbous or saccate genus Mohavea are solitary bees in the genera Antirrhinum (Sutton 1988). Xeralictus (Halictidae) and Hesperapis (Melit- The palate is a structure formed by the tidae), which are the primary pollinators of arching of the bottom of the corolla-tube Mentzelia (Little 1983). upwards to close the entrance. Other than in The unique floral features and the cyathi- the Antirrhineae, a palate is known to be form seed structure encouraged several present in some members of Cheloneae and authors (Rothmaler 1943, Speta 1982, Elisens Gratioleae (Pennell 1935, Kampany 1995). In 1985c) to treat Mohavea as a subtribe of its certain members of Antirrhineae, e.g. Linaria, own. Despite these unique features, Mohavea Antirrhinum, Asarina, and Maurandella, the in the present analyses is nested within the palate is conspicuous and completely blocks Antirrhinum clade (Figs. 2, 3). In the analyses the corolla-tube, whereas in Anarrhinum, Mohavea is placed in a position with Howel- Kickxia and Chaenorhinum the arching is liella as a sister taxon. Howelliella has a low and does not block the mouth of the tube. poorly developed palate and widely gaping Several of the New World Antirrhineae, throat with two prominent longitudinal folds including Rhodochiton, Lophospermum, and in its floor. It is reported to be pollinated by Maurandya, have flowers that are relatively hummingbirds as well as by bees (Thompson large, more or less actinomorphic, and without 1988). a palate. Many of them have been reported to 236 M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) be pollinated by hummingbirds (Elisens scribed by Sutton (1988), appears to be 1986). Although bee-pollination is most com- monophyletic. mon and probably ancestral in the Antir- The Gambelia clade. The clade is com- rhineae, the present results indicate that posed of a small but taxonomically complex hummingbird-pollination has obviously evolved group in the New World historically variously independently at least three times, in the treated as the three genera Galvezia, Gambe- Maurandya, Gambelia, and Antirrhinum lia, and Saccularia as by Rothmaler (1943), as clades (Fig. 4). one genus Galvezia as by Munz (1926), or as a Comparison with previous subtribal section of Maurandya or Antirrhinum as by classification. The present results though Bentham (1876), and Wettstein (1891). In his differing in many ways have some similarities subtribal classification Rothmaler (1943) trea- with Rothmaler's classification. His treatment ted the group as a separate subtribe Gambe- of Mohaveinae and Anarrhininae as mono- liinae. Recently Sutton (1988) recognised two typic subtribes (Rothmaler 1943) is not genera, Galvezia in South America and supported by the present results. The results Gambelia in North America. He also pointed are also incongruent with the circumscriptions out that Galvezia is unique in the tribe in of the subtribe Maurandyinae made by having a totally southern hemispheric distribu- Rothmaler (1943) and Elisens (1985a), and tion, but stressed its close relationship with the subtribes Linariinae and Antirrhininae as Gambelia. Elisens and Tomb (1983) stated circumscribed by Speta (1982). No formal that it is difficult to draw any conclusions subtribal classification is proposed here, but regarding the inter- or infrageneric classifica- some further comments on the four clades are tion of these two taxa using seed morphology. given below, including the placing of all In the present analyses Galvezia and Gambelia genera recognised by Sutton (1988). come out as sister taxa with a bootstrap support The Anarrhinum ¢lade. In his subtribal of 100. The molecular differences detected classification Rothmaler (1943) was unable to between the genera are very small, which may group Anarrhinum with any other genus and support the view that they should be treated as therefore placed it in a subtribe of its own, one widely circumscribed genus. All members Anarrhininae. The present results show that of the clade have a shrubby habit and are the Old World genera Anarrhinum and Kickxia unique in possessing flowers with long narrow are sister taxa. Anarrhinum is similar to corolla-tubes pollinated by hummingbirds Kickxia in its unusual arrangement of the (Fig. 4). anthers, dehiscence of the capsule, position of The Maurandya clade. The Maurandyinae the hilum, and in its ornamentation of the as delimited by Rothmaler (1943) included seeds (Sutton .1988). The close relationship only New World Antirrhineae. On the basis of between Anarrhinum and Kickxia is also similarities in foliage and floral characters supported by the similarity of the protein Pennell (1947) concluded that Asarina and bodies in the cell nuclei (Speta 1979). The Maurandya are congeneric. Elisens (1985a) present results show that the Anarrhinum clade rejected Pennell's view and argued that the is strongly supported and is characterised two taxa are very distinct in many ways, above all by three unique synapomorphies, including different chromosome numbers different shapes of upper and lower leaves, (Asarina x=9 and Maurandya x= 12) and connate anthers forming a ring, and median geographic distribution. position of hilum. The present results further The present results indicate that Asarina indicate that the group is sister to the rest of and Cymbalaria are sister taxa to the New the Antirrhineae. The two sections of Kickxia World Maurandyinae and suggest that the are sister groups and the genus, as circum- subtribe Maurandyinae sensu Elisens could M. Ghebrehiwet et al.: Phylogeny of the tribe Antirrhineae (Scrophulariaceae) 237 well be expanded further to include both A number of other segregate genera of Asarina and Cymbalaria. Antirrhinum, Linaria, and Chaenorhinum, and As can be deduced from the morphologi- which, like Neogaerrhinum, Pseudorontium, cal tree (Fig. 1) some further genera that and Sairocarpus, were not sampled for the probably belong to the Maurandya clade, but present molecular analysis are Acanthorrhi- which were not included in the molecular hum, Albraunia, and Holzneria, in the Old analysis, are Holmgrenanthe, Mabrya, Lophos- World, and Nuttallanthus in the New World. permum, and Epixiphium. All members of this They agree in various characters with either group are suffrutescent herbs with procumbent Antirrhinum, Linaria, or Chaenorhinum, and or climbing stems and have orbiculate or probably all belong to the Antirrhinum clade. deltate leaf-blades with palmate venation. We thank Mattias Iwarsson, the Botanical The Antirrhinum clade. This clade largely Garden of Uppsala University, and Hans V. corresponds to the subtribe Linariinae sensu Hansen, the Botanical Garden of the University Rothmaler (1943, 1954), which includes of Copenhagen, for providing seeds, and Roland among others Linaria, Antirrhinum, Chaenor- Moberg, UPS, for access to herbarium material. hinum and their respective segregate genera. Christina Grusell is acknowledged for help with On the basis of chromosome numbers Speta the greenhouse work, Nahid Heidari for sequen- (1982) delimited Linariinae only to Linaria cing 12 of the taxa, and Anders Nilsson for with x--6 and proposed a new subtribe, valuable information on literature on pollination. Antirrhininae, for the remaining genera with We also thank K~re Bremer and Bengt Oxelman x = 7, 8 and 9. The present results show that for constructive comments on the manuscript. The both Linaria and Antirrhinum belong to a study was supported by a scholarship from Sida/ SAREC (Swedish International Development clade that contains both the Old World genera Agency/Swedish Agency for Research Co-opera- Chaenorhinum, Linaria, Antirrhinum, Miso- tion with Developing Countries), and a grant from pates, and Schweinfurthia, and the New World the Swedish Natural Science Research Council to genera Howelliella and Mohavea. The clade is Birgitta Bremer. well supported in the molecular analysis with a bootstrap value of 80, but this is slightly reduced to 74 in the combined analysis. References Among the genera that are included in Armbruster W. S. (1992) Phylogeny and evolution Linariinae by Rothmaler (1943) or in Antir- of plant-animal interactions. Bioscience 42: 12- rhininae by Speta (1982) are Asarina, Cym- 20. balaria and Kickxia, but this is not compatible Armbmster W. S. (1993) Evolution of plant with the present results. 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